scholarly journals Direct digital sensing of proteins in solution through single-molecule optofluidics

2020 ◽  
Author(s):  
Georg Krainer ◽  
Kadi L. Saar ◽  
William E. Arter ◽  
Raphaël P.B. Jacquat ◽  
Quentin Peter ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Detection ◽  
Single Step ◽  
Surface Immobilization ◽  
Biomedical Analysis ◽  
Affinity Reagents ◽  
Probe Concentration ◽  
Probe Affinity ◽  
Increased Sensitivity ◽  
Highly Sensitive Detection

AbstractHighly sensitive detection of proteins is of central importance to biomolecular analysis and diagnostics. Conventional protein sensing assays, such as ELISAs, remain reliant on surface-immobilization of target molecules and multi-step washing protocols for the removal of unbound affinity reagents. These features constrain parameter space in assay design, resulting in fundamental limitations due to the underlying thermodynamics and kinetics of the immunoprobe–analyte interaction. Here, we present a new experimental paradigm for the quantitation of protein analytes through the implementation of an immunosensor assay that operates fully in solution and realizes rapid removal of excess probe prior to detection without the need of washing steps. Our single-step optofluidic approach, termed digital immunosensor assay (DigitISA), is based on microfluidic electrophoretic separation combined with single-molecule laser-induced fluorescence microscopy and enables calibration-free in-solution protein detection and quantification within seconds. Crucially, the solution-based nature of our assay and the resultant possibility to use arbitrarily high probe concentrations combined with its fast operation timescale enables quantitative binding of analyte molecules regardless of the capture probe affinity, opening up the possibility to use relatively weak-binding affinity reagents such as aptamers. We establish and validate the DigitISA platform by probing a biomolecular biotin–streptavidin binding complex and demonstrate its applicability to biomedical analysis by quantifying IgE–aptamer binding. We further use DigitISA to detect the presence of α-synuclein fibrils, a biomarker for Parkinson’s disease, using a low-affinity aptamer at high probe concentration. Taken together, DigitISA presents a fundamentally new route to surface-free specificity, increased sensitivity, and reduced complexity in state-of-the-art protein detection and biomedical analysis.

Highly Sensitive Detection of Paraquat with Pillar[5]arene as an aptamer in α-Hemolysin Nanopore

2021 ◽  
Author(s):  
Xiaojia Jiang ◽  
Mingsong Zang ◽  
Fei Li ◽  
Chunxi Hou ◽  
Quan Luo ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Single Molecule Detection ◽  
Sensitive Detection ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
The Real ◽  
Highly Sensitive ◽  
Highly Sensitive Detection

Biological nanopore-based techniques have attracted more and more attention recently in the field of single-molecule detection, because they allow the real-time, sensitive, high-throughput analysis. Herein, we report an engineered biological...

scholarly journals Nanopore Technology for the Application of Protein Detection

Nanomaterials ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1942
Author(s):  
Xiaoqing Zeng ◽  
Yang Xiang ◽  
Qianshan Liu ◽  
Liang Wang ◽  
Qianyun Ma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Detection ◽  
High Sensitivity ◽  
Single Molecule Detection ◽  
Sequence Detection ◽  
Fast Detection ◽  
Material Basis ◽  
Sensing Technology ◽  
Detection Technology ◽  
Structure Recognition

Protein is an important component of all the cells and tissues of the human body and is the material basis of life. Its content, sequence, and spatial structure have a great impact on proteomics and human biology. It can reflect the important information of normal or pathophysiological processes and promote the development of new diagnoses and treatment methods. However, the current techniques of proteomics for protein analysis are limited by chemical modifications, large sample sizes, or cumbersome operations. Solving this problem requires overcoming huge challenges. Nanopore single molecule detection technology overcomes this shortcoming. As a new sensing technology, it has the advantages of no labeling, high sensitivity, fast detection speed, real-time monitoring, and simple operation. It is widely used in gene sequencing, detection of peptides and proteins, markers and microorganisms, and other biomolecules and metal ions. Therefore, based on the advantages of novel nanopore single-molecule detection technology, its application to protein sequence detection and structure recognition has also been proposed and developed. In this paper, the application of nanopore single-molecule detection technology in protein detection in recent years is reviewed, and its development prospect is investigated.

scholarly journals A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Jasper H. M. van der Velde ◽  
Jens Oelerich ◽  
Jingyi Huang ◽  
Jochem H. Smit ◽  
Atieh Aminian Jazi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Unnatural Amino Acids ◽  
Photophysical Properties ◽  
Super Resolution ◽  
Single Step ◽  
General Strategy ◽  
Self Healing ◽  
Chemical Transformations ◽  
Scaffolding Strategy ◽  
Organic Fluorophore

Abstract Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with ‘self-healing’ properties. To date, utilization of such fluorophore derivatives is rare due to their elaborate multi-step synthesis. Here we present a general strategy to covalently link a synthetic organic fluorophore simultaneously to a photostabilizer and biomolecular target via unnatural amino acids. The modular approach uses commercially available starting materials and simple chemical transformations. The resulting photostabilizer–dye conjugates are based on rhodamines, carbopyronines and cyanines with excellent photophysical properties, that is, high photostability and minimal signal fluctuations. Their versatile use is demonstrated by single-step labelling of DNA, antibodies and proteins, as well as applications in single-molecule and super-resolution fluorescence microscopy. We are convinced that the presented scaffolding strategy and the improved characteristics of the conjugates in applications will trigger the broader use of intramolecular photostabilization and help to emerge this approach as a new gold standard.

scholarly journals Leveraging Surface Siloxide Electronics to Enhance Relaxation Properties of a Single-Molecule Magnet

2020 ◽  
Author(s):  
Maciej Korzynski ◽  
Zachariah Berkson ◽  
Boris Le Guennic ◽  
Olivier Cador ◽  
Christophe Copéret
Keyword(s):  
Single Molecule ◽  
Relaxation Times ◽  
Device Fabrication ◽  
Information Storage ◽  
Commercial Application ◽  
Surface Immobilization ◽  
Single Molecule Magnets ◽  
Magnetization Relaxation ◽  
Surface Deposition ◽  
Magnitude Increase

Single-molecule magnets (SMMs) hold promise for unmatched information storage density as well as applications in quantum computing and spintronics. To date, the most successful SMMs are organometallic lanthanide complexes. However, their surface immobilization, one of the requirements for device fabrication and commercial application, remains challenging due to sensitivity of magnetic properties to small changes in the electronic structure of the parent SMM. Thus, finding controlled approaches to SMM surface deposition is a timely challenge. In this contribution we apply the concept of isolobality to identify siloxides present at the surface of partially dehydroxylated silica as a suitable replacement for archetypal ligand architectures in organometallic SMMs. We demonstrate theoretically and experimentally that isolated siloxide anchorages not only enable successful immobilization, but also lead to two-orders-of-magnitude increase in magnetization relaxation times and provide magnetic site dilution.

scholarly journals Streamlined circular proximity ligation assay provides high stringency and compatibility with low-affinity antibodies

2018 ◽  
Vol 115 (5) ◽  
pp. E925-E933 ◽  
Author(s):  
Roxana Jalili ◽  
Joe Horecka ◽  
James R. Swartz ◽  
Ronald W. Davis ◽  
Henrik H. J. Persson
Keyword(s):  
Limit Of Detection ◽  
Biological Fluids ◽  
Protein Detection ◽  
Ease Of Use ◽  
Proximity Ligation Assay ◽  
Quantitative Detection ◽  
High Signal ◽  
Affinity Reagents ◽  
Proximity Ligation ◽  
Assay Format

Proximity ligation assay (PLA) is a powerful tool for quantitative detection of protein biomarkers in biological fluids and tissues. Here, we present the circular proximity ligation assay (c-PLA), a highly specific protein detection method that outperforms traditional PLA in stringency, ease of use, and compatibility with low-affinity reagents. In c-PLA, two proximity probes bind to an analyte, providing a scaffolding that positions two free oligonucleotides such that they can be ligated into a circular DNA molecule. This assay format stabilizes antigen proximity probe complexes and enhances stringency by reducing the probability of random background ligation events. Circle formation also increases selectivity, since the uncircularized DNA can be removed enzymatically. We compare this method with traditional PLA on several biomarkers and show that the higher stringency for c-PLA improves reproducibility and enhances sensitivity in both buffer and human plasma. The limit of detection ranges from femtomolar to nanomolar concentrations for both methods. Kinetic analyses using surface plasmon resonance (SPR) and biolayer interferometry (BLI) reveal that the variation in limit of detection is due to the variation in antibody affinity and that c-PLA outperforms traditional PLA for low-affinity antibodies. The lower background signal can be used to increase proximity probe concentration while maintaining a high signal-to-noise ratio, thereby enabling the use of low-affinity reagents in a homogeneous assay format. We anticipate that the advantages of c-PLA will be useful in a variety of clinical protein detection applications where high-affinity reagents are lacking.

scholarly journals Ultrasensitive Detection of Clostridioides difficile Toxins in Stool by Use of Single-Molecule Counting Technology: Comparison with Detection of Free Toxin by Cell Culture Cytotoxicity Neutralization Assay

2019 ◽  
Vol 57 (11) ◽  
Author(s):  
Glen Hansen ◽  
Stephen Young ◽  
Alan H. B. Wu ◽  
Emily Herding ◽  
Vickie Nordberg ◽  
...  
Keyword(s):  
Cell Culture ◽  
Single Molecule ◽  
Neutralization Assay ◽  
Single Step ◽  
Multicenter Studies ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Ultrasensitive Assay ◽  
A Cell ◽  
Positive Agreement

ABSTRACT Laboratory tests for Clostridioides difficile infection (CDI) rely on the detection of free toxin or molecular detection of toxin genes. The Singulex Clarity C. diff toxins A/B assay is a rapid, automated, and ultrasensitive assay that detects C. difficile toxins A and B in stool. We compared CDI assays across two prospective multicenter studies to set a cutoff for the Clarity assay and to independently validate the performance compared with that of a cell culture cytotoxicity neutralization assay (CCCNA). The cutoff was set by two sites testing fresh samples from 897 subjects with suspected CDI and then validated at four sites testing fresh samples from 1,005 subjects with suspected CDI. CCCNA testing was performed at a centralized laboratory. Samples with discrepant results between the Clarity assay and CCCNA were retested with CCCNA when the Clarity result agreed with that of at least one comparator method; toxin enzyme immunoassays (EIA), glutamate dehydrogenase (GDH) detection, and PCR were performed on all samples. The cutoff for the Clarity assay was set at 12.0 pg/ml. Compared to results with CCCNA, the Clarity assay initially had 85.2% positive agreement and 92.4% negative agreement. However, when samples with discrepant results between the Clarity assay and CCCNA in the validation study were retested by CCCNA, 13/17 (76.5%) Clarity-negative but CCCNA-positive samples (Clarity+/CCCNA−) became CCCNA−, and 5/26 (19.2%) Clarity+/CCCNA− samples became CCCNA+, resulting in a 96.3% positive agreement and 93.0% negative agreement between Clarity and CCCNA results. The toxin EIA had 59.8% positive agreement with CCCNA. The Clarity assay was the most sensitive free-toxin immunoassay, capable of providing CDI diagnosis in a single-step solution. A different CCCNA result was reported for 42% of retested samples, increasing the positive agreement between Clarity and CCCNA from 85.2% to 96.3% and indicating the challenges of comparing free-toxin results to CCCNA results as a reference standard.

scholarly journals 1088. Ultrasensitive Detection of C. difficile Toxins in Stool Using Single Molecule Counting Technology: A Multicenter Study for Evaluation of Clinical Performance

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S325-S326 ◽  
Author(s):  
Stephen Young ◽  
Ray Mills ◽  
Christen Griego-Fullbright ◽  
Aaron Wagner ◽  
Emily Herding ◽  
...  
Keyword(s):  
Single Molecule ◽  
Multicenter Study ◽  
Clinical Performance ◽  
Turnaround Time ◽  
Neutralization Assay ◽  
Single Step ◽  
Reporting Structure ◽  
One Step ◽  
Automated Immunoassay ◽  
Higher Sensitivity

Abstract Background Commercially available tests for Clostridium difficile infection (CDI) make test selection by the laboratory difficult due to the following unsatisfactory characteristics: long turnaround time, poor sensitivity, and/or poor specificity. The Singulex Clarity® C. diff toxins A/B assay (in development) is a rapid and automated immunoassay for the detection of C. difficile toxins A and B in stool, with analytical limits of detection for toxins A and B at 2.0 and 0.7 pg/mL, respectively. In this multicenter study, the clinical performance of the Singulex Clarity C. diff toxins A/B assay was compared with standalone PCR, a multistep algorithm with enzyme immunoassay (EIA) and PCR, and cell cytotoxicity neutralization assay (CCNA). Methods Fresh samples from 267 subjects with suspected CDI were tested at two sites (Minneapolis Medical Research Foundation and TriCore Reference Laboratories) with the Singulex Clarity assay, PCR (Xpert®C. difficile), and EIA (C. Diff Quik Chek Complete®) for GDH and toxin testing. The performance of the assays and multistep algorithms were evaluated against CCNA (Microbiology Specialists, Inc.). Results The overall CDI prevalence was 15.7%. The Singulex Clarity C. diff toxins A/B assay had 90.5% sensitivity and 96.0% specificity, with a 98.2% negative predictive value when compared with CCNA, and the Clarity assay’s AuROC was 0.9534. PCR had 90.5% sensitivity and 91.1% specificity. Compared with CCNA, the toxin EIA had 47.6% sensitivity and 100% specificity. Testing with a multistep algorithm using EIA with discordant results reflexed to PCR resulted in 85.7% sensitivity and 94.7% specificity. Conclusion The ultrasensitive Singulex Clarity C. diff toxins A/B assay is equivalent to the sensitivity of PCR while providing higher specificity. Compared with a multistep algorithm, the Clarity assay provides higher sensitivity and specificity while providing faster time-to-result in a simpler-to-understand, one-step reporting structure, allowing for a standalone, single-step solution for detection of C. difficile toxins in patients with suspected CDI. Disclosures E. Friedland, Singulex, Inc.: Employee, Salary. A. Bartolome, Singulex, Inc.: Employee, Salary. A. Almazan, Singulex, Inc.: Employee, Salary. S. Tam, Singulex, Inc.: Employee, Salary. S. Biscocho, Singulex, Inc.: Employee, Salary. S. Abusali, Singulex, Inc.: Employee, Salary. J. Sandlund, Singulex, Inc.: Employee, Salary. J. Estis, Singulex, Inc.: Employee, Salary. J. Bishop, Singulex, Inc.: Employee, Salary.

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