Water-developable negative-tone single-molecule resists: high-sensitivity nonchemically amplified resists

DNA lesion such as metholcytosine(mC), 8-OXO-guanine(OG), inosine(I) etc could cause the genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remain challenge due to the massive varieties and less distinguishable readouts for minor structural variations. Moreover, standard amplification and labelling hardly works in DNA lesions detection. Herein, we designed a single molecule interface from the mutant K238Q Aerolysin, whose confined sensing region shows the high compatible to capture and then directly convert each base lesion into distinguishable current readouts. Compared with previous single molecule sensing interface, the resolution of the K238Q Aerolysin nanopore is enhanced by 2-order. The novel K238Q could direct discriminate at least 3 types (mC, OG, I) lesions without lableing and quantify modification sites under mixed hetero-composition condition of oligonucleotide. Such nanopore could be further applied to diagnose genetic diseases at high sensitivity.

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Nanopore Technology for the Application of Protein Detection

Nanomaterials ◽

10.3390/nano11081942 ◽

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.

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Single-copy detection of somatic variants from solid and liquid biopsy

Scientific Reports ◽

10.1038/s41598-021-85545-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ana-Luisa Silva ◽

Paulina Klaudyna Powalowska ◽

Magdalena Stolarek ◽

Eleanor Ruth Gray ◽

Rebecca Natalie Palmer ◽

...

Keyword(s):

Real Time ◽

Single Molecule ◽

Real Time Pcr ◽

Clinical Decision Making ◽

High Sensitivity ◽

Clinical Decision ◽

Single Copy ◽

Turnaround Time ◽

Copy Detection ◽

Allele Specific

AbstractAccurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

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High sensitivity optical microscope for single molecule spectroscopy studies

Review of Scientific Instruments ◽

10.1063/1.1777385 ◽

2004 ◽

Vol 75 (8) ◽

pp. 2746-2751 ◽

Cited By ~ 11

Author(s):

Gabriele Malengo ◽

Roberto Milani ◽

Fabio Cannone ◽

Silke Krol ◽

Alberto Diaspro ◽

...

Keyword(s):

Single Molecule ◽

High Sensitivity ◽

Optical Microscope ◽

Single Molecule Spectroscopy ◽

Spectroscopy Studies

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Scanning single-molecule counting system for Eprobe with highly simple and effective approach

PLoS ONE ◽

10.1371/journal.pone.0243319 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0243319

Author(s):

Takeshi Hanami ◽

Tetsuya Tanabe ◽

Takuya Hanashi ◽

Mitsushiro Yamaguchi ◽

Hidetaka Nakata ◽

...

Keyword(s):

Nucleic Acid ◽

Single Molecule ◽

Detection System ◽

Signal To Noise Ratio ◽

High Sensitivity ◽

Nucleic Acid Detection ◽

Digital Measurement ◽

Excitation Light ◽

Target Nucleic Acid ◽

Fluorescent Molecules

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

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Single-Molecule Detection of DNA in a Nanochannel by High-Field Strength-Assisted Electrical Impedance Spectroscopy

Micromachines ◽

10.3390/mi10030189 ◽

2019 ◽

Vol 10 (3) ◽

pp. 189 ◽

Cited By ~ 1

Author(s):

Porpin Pungetmongkol ◽

Takatoki Yamamoto

Keyword(s):

Single Molecule ◽

High Intensity ◽

High Sensitivity ◽

Electrical Impedance Spectroscopy ◽

Random Coil ◽

Label Free ◽

Electrode Gap ◽

Label Free Detection ◽

Intensity Field ◽

Nanogap Electrodes

Many researchers have fabricated micro and nanofluidic devices incorporating optical, chemical, and electrical detection systems with the aim of achieving on-chip analysis of macromolecules. The present study demonstrates a label-free detection of DNA using a nanofluidic device based on impedance measurements that is both sensitive and simple to operate. Using this device, the electrophoresis and dielectrophoresis effect on DNA conformation and the length dependence were examined. A low alternating voltage was applied to the nanogap electrodes to generate a high intensity field (>0.5 MV/m) under non-faradaic conditions. In addition, a 100 nm thick gold electrode was completely embedded in the substrate to allow direct measurements of a solution containing the sample passing through the gap, without any surface modification required. The high intensity field in this device produced a dielectrophoretic force that stretched the DNA molecule across the electrode gap at a specific frequency, based on back and forth movements between the electrodes with the DNA in a random coil conformation. The characteristics of 100 bp, 500 bp, 1 kbp, 5 kbp, 10 kbp, and 48 kbp λ DNA associated with various conformations were quantitatively analyzed with high resolution (on the femtomolar level). The sensitivity of this system was found to be more than about 10 orders of magnitude higher than that obtained from conventional linear alternating current (AC) impedance for the analysis of bio-polymers. This new high-sensitivity process is expected to be advantageous with regard to the study of complex macromolecules and nanoparticles.

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Blood-based high sensitivity measurements of beta-amyloid and phosphorylated tau as biomarkers of Alzheimer’s disease: a focused review on recent advances

Journal of Neurology Neurosurgery & Psychiatry ◽

10.1136/jnnp-2021-327370 ◽

2021 ◽

pp. jnnp-2021-327370

Author(s):

Joyce R. Chong ◽

Nicholas J. Ashton ◽

Thomas K. Karikari ◽

Tomotaka Tanaka ◽

Michael Schöll ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Single Molecule ◽

Emerging Technologies ◽

Amyloid Β ◽

High Sensitivity ◽

Phosphorylated Tau ◽

Meso Scale Discovery ◽

Aβ Peptides

Discovery and development of clinically useful biomarkers for Alzheimer’s disease (AD) and related dementias have been the focus of recent research efforts. While cerebrospinal fluid and positron emission tomography or MRI-based neuroimaging markers have made the in vivo detection of AD pathology and its consequences possible, the high cost and invasiveness have limited their widespread use in the clinical setting. On the other hand, advances in potentially more accessible blood-based biomarkers had been impeded by lack of sensitivity in detecting changes in markers of the hallmarks of AD, including amyloid-β (Aβ) peptides and phosphorylated tau (P-tau). More recently, however, emerging technologies with superior sensitivity and specificity for measuring Aβ and P-tau have reported high concordances with AD severity. In this focused review, we describe several emerging technologies, including immunoprecipitation-mass spectrometry (IP-MS), single molecule array and Meso Scale Discovery immunoassay platforms, and appraise the current literature arising from their use to identify plaques, tangles and other AD-associated pathology. While there is potential clinical utility in adopting these technologies, we also highlight the further studies needed to establish Aβ and P-tau as blood-based biomarkers for AD, including validation with existing large sample sets, new independent cohorts from diverse backgrounds as well as population-based longitudinal studies. In conclusion, the availability of sensitive and reliable measurements of Aβ peptides and P-tau species in blood holds promise for the diagnosis, prognosis and outcome assessments in clinical trials for AD.

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645. Singulex Clarity Norovirus Assay (In Development) Provides Ultrasensitive Detection of Norovirus Genogroups I and II

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.713 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S297-S298

Author(s):

Gipshu Dave ◽

Phoebe Katzenbach ◽

Johanna Sandlund ◽

Joel Estis ◽

Ali Mukherjee ◽

...

Keyword(s):

Single Molecule ◽

Operating Characteristic ◽

High Sensitivity ◽

Excellent Performance ◽

C Statistic ◽

Stool Samples ◽

Capture Antibody ◽

Automated Platform ◽

Paramagnetic Microparticles ◽

Detection Reagent

Abstract Background Commercially available enzyme immunoassays (EIAs) for detection of norovirus antigen have poor sensitivity and are limited to use in investigations of a gastroenteritis outbreak. Hence, there remains a need for a standalone high-sensitivity assay that enables rapid and accurate detection of norovirus antigen. Methods The Singulex Clarity norovirus assay is currently in development for use on the Singulex Clarity® system (Singulex Inc., Alameda, CA, USA), a fully-automated platform powered by Single Molecule Counting technology (registered with the FDA and CE marked). The assay uses paramagnetic microparticles bound to capture antibody and a fluorescently labeled reporter antibody to detect virion capsid protein of norovirus genogroups I (GI) and II (GII) in the stool. For the development of Clarity Norovirus assay, diagnostic performance of 4 antibody pairs (as Capture and Detection reagent) were evaluated by testing 137 stool samples from patients with suspected norovirus infection. Samples were sourced from three providers: (1) 90 genotyped samples of which 75 were positive (19 different genotypes) and 15 were negative by the CDC assay, (2) 3 samples positive and 5 samples negative by the BioFire® FilmArray® Gastrointestinal Panel, and (3) 39 samples negative by a lab-developed test using Cepheid reagents (SmartCycler®). Results From all the antibody pairs tested, one of the pairs had best performance with the area under the receiver operating characteristic (AuROC) curve demonstrating a C-Statistic of 0.959 (95% CI 0.921–0.997), compared with AuROC C-statistic of 0.943 (95% CI 0.896–0.990), 0.871 (95% CI 0.807–0.936), and 0.914 (95% CI 0.863–0.964) for the three other pairs. The Clarity assay detected all 19 different genotypes tested (figures). Conclusion The ultrasensitive and rapid Clarity norovirus assay (in development) for detection of GI and GII demonstrated excellent performance with one of the antibody pairs tested and detected all 19 tested genotypes. The Clarity assay may offer a standalone solution for norovirus diagnostics. Disclosures All authors: No reported disclosures.

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Application of Solid-State Nanopore in Protein Detection

International Journal of Molecular Sciences ◽

10.3390/ijms21082808 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2808 ◽

Cited By ~ 2

Author(s):

Yuhan Luo ◽

Linlin Wu ◽

Jing Tu ◽

Zuhong Lu

Keyword(s):

Solid State ◽

Chemical Modification ◽

Single Molecule ◽

Protein Detection ◽

High Sensitivity ◽

Protein Sequencing ◽

Its Sequence ◽

Sequence Structure ◽

Single Molecule Level ◽

Protein Molecules

A protein is a kind of major biomacromolecule of life. Its sequence, structure, and content in organisms contains quite important information for normal or pathological physiological process. However, research of proteomics is facing certain obstacles. Only a few technologies are available for protein analysis, and their application is limited by chemical modification or the need for a large amount of sample. Solid-state nanopore overcomes some shortcomings of the existing technology, and has the ability to detect proteins at a single-molecule level, with its high sensitivity and robustness of device. Many works on detection of protein molecules and discriminating structure have been carried out in recent years. Single-molecule protein sequencing techniques based on solid-state nanopore are also been proposed and developed. Here, we categorize and describe these efforts and progress, as well as discuss their advantages and drawbacks.

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