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

2021 ◽  
Author(s):  
Qiang Zeng ◽  
Xiaoyan Zhou ◽  
Yuting Yang ◽  
Yi Sun ◽  
Jingan Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Specific Binding ◽  
Single Molecules ◽  
Amyloid Β ◽  
Molecular Complexes ◽  
Biomarker Detection ◽  
Complex Samples ◽  
Practical Applications

Abstract 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.

scholarly journals A Sensitive and Cost-Effective Chemiluminescence ELISA for Measurement of Amyloid-β 1-42 Peptide in Human Plasma

2020 ◽  
Vol 78 (3) ◽  
pp. 1237-1244
Author(s):  
Pankaj D. Mehta ◽  
Bruce A. Patrick ◽  
David L. Miller ◽  
Patricia K. Coyle ◽  
Thomas Wisniewski
Keyword(s):  
Human Plasma ◽  
Single Molecule ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Amyloid Β ◽  
Cost Effective ◽  
Measurement Range ◽  
Rabbit Monoclonal Antibody ◽  
Low Levels ◽  
The Brain

Background: Amyloid-β42 (Aβ42) is associated with plaque formation in the brain of patients with Alzheimer’s disease (AD). Studies have suggested the potential utility of plasma Aβ42 levels in the diagnosis, and in longitudinal study of AD pathology. Conventional ELISAs are used to measure Aβ42 levels in plasma but are not sensitive enough to quantitate low levels. Although ultrasensitive assays like single molecule array or immunoprecipitation-mass spectrometry have been developed to quantitate plasma Aβ42 levels, the high cost of instruments and reagents limit their use. Objective: We hypothesized that a sensitive and cost-effective chemiluminescence (CL) immunoassay could be developed to detect low Aβ42 levels in human plasma. Methods: We developed a sandwich ELISA using high affinity rabbit monoclonal antibody specific to Aβ42. The sensitivity of the assay was increased using CL substrate to quantitate low levels of Aβ42 in plasma. We examined the levels in plasma from 13 AD, 25 Down syndrome (DS), and 50 elderly controls. Results: The measurement range of the assay was 0.25 to 500 pg/ml. The limit of detection was 1 pg/ml. All AD, DS, and 45 of 50 control plasma showed measurable Aβ42 levels. Conclusion: This assay detects low levels of Aβ42 in plasma and does not need any expensive equipment or reagents. It offers a preferred alternative to ultrasensitive assays. Since the antibodies, peptide, and substrate are commercially available, the assay is well suited for academic or diagnostic laboratories, and has a potential for the diagnosis of AD or in clinical trials.

scholarly journals Improved immunoassay sensitivity and specificity using single-molecule colocalization

2021 ◽  
Author(s):  
Amani A. Hariri ◽  
Sharon S. Newman ◽  
Steven Tan ◽  
Dan Mamerow ◽  
Michael Eisenstein ◽  
...  
Keyword(s):  
Single Molecule ◽  
Total Internal Reflection ◽  
Limit Of Detection ◽  
Specific Binding ◽  
Heterogeneous Distribution ◽  
Tnf Α ◽  
Target Molecules ◽  
Series Of Experiments ◽  
Consistent Performance ◽  
True Signal

Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still suffers some notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background arising from the interaction of detection antibodies with the assay substrate or interferents in the sample matrix. Single-Molecule Colocalization Assay (SiMCA) overcomes this problem by employing total internal reflection fluorescence (TIRF) microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized fluorescent signals, we can essentially eliminate the confounding effects of background produced by non-specific binding of detection antibodies. We further employed a normalization strategy to account for the heterogeneous distribution of the capture antibodies, greatly improving the reproducibility of our measurements. In a series of experiments with TNF-α, we show that SiMCA can achieve a three-fold lower limit of detection compared to conventional single-color assays using the same antibodies and exhibits consistent performance for assays performed in complex specimens such as chicken serum and human blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects.

scholarly journals Ultra-rapid and highly efficient enrichment of organic pollutants via magnetic mesoporous nanosponge for ultrasensitive nanosensors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lingling Zhang ◽  
Yu Guo ◽  
Rui Hao ◽  
Yafei Shi ◽  
Hongjun You ◽  
...  
Keyword(s):  
Organic Pollutants ◽  
Single Molecule ◽  
Cross Sections ◽  
Limit Of Detection ◽  
Detection Sensitivity ◽  
Practical Applications ◽  
Single Molecule Level ◽  
Surface Enhanced Raman ◽  
Low Sensitivity ◽  
Enrichment Strategy

AbstractCurrently, owing to the single-molecule-level sensitivity and highly informative spectroscopic characteristics, surface-enhanced Raman scattering (SERS) is regarded as the most direct and effective detection technique. However, SERS still faces several challenges in its practical applications, such as the complex matrix interferences, and low sensitivity to the molecules of intrinsic small cross-sections or weak affinity to the surface of metals. Here, we show an enrichment-typed sensing strategy with both excellent selectivity and ultrahigh detection sensitivity based on a powerful porous composite material, called mesoporous nanosponge. The nanosponge consists of porous β-cyclodextrin polymers immobilized with magnetic NPs, demonstrating remarkable capability of effective and fast removal of organic micropollutants, e.g., ~90% removal efficiency within ~1 min, and an enrichment factor up to ~103. By means of this current enrichment strategy, the limit of detection for typical organic pollutants can be significantly improved by 2~3 orders of magnitude. Consequently, the current enrichment strategy is proved to be applicable in a variety of fields for portable and fast detection, such as Raman and fluorescent sensing.

Digital Detection of Nanoparticles: Viral Diagnostics and Multiplexed Protein and Nucleic Acid Assays

2015 ◽  
Vol 1720 ◽  
Author(s):  
M. S. Ünlü
Keyword(s):  
Limit Of Detection ◽  
Simultaneous Detection ◽  
Detection Sensitivity ◽  
Optical Antenna ◽  
Dna Arrays ◽  
Complex Samples ◽  
Practical Applications ◽  
Nanoparticle Detection ◽  
Nucleic Acid Assays ◽  
Imaging Sensor

ABSTRACTSynthetic nanoparticles have made significant impact across a broad range of technological applications including optical nanoantennas, ultra-sensitive imaging and sensing, and diagnostics and therapeutics. Natural nanoparticles such as viruses and pollutants are major concerns for human health. High-throughput characterization of nanoparticles in terms of their size and shape is crucial for practical applications of synthetic nanoparticles and highly sensitive pathogen identification. Recently, we have demonstrated Interferometric Reflectance Imaging Sensor (IRIS) with the ability to detect single nanoscale particles [1,2].In single-particle modality of IRIS (SP-IRIS), the interference of light reflected from the sensor surface is modified by the presence of particles producing a distinct signal that reveals the size of the particle. In our approach, the dielectric layered structure acts as an optical antenna optimizing the elastic scattering characteristics of nanoparticles for sensitive detection and analysis. We have demonstrated identification of virus articles in complex samples for various viruses in multiplexed format. Size discrimination of the imaged nanoparticles (virions) allows differentiation between modified viruses having different genome lengths and facilitates a reduction in the counting of non-specifically bound particles to achieve a limit-of-detection (LOD) of 5x103 pfu/mL for the Ebola and Marburg VSV pseudotypes. We have demonstrated the simultaneous detection of multiple viruses in serum or whole blood as well as in samples contaminated with high levels of bacteria [3]. Single nanoparticle detection with IRIS has shown promising results for protein [4] and DNA arrays with attomolar detection sensitivity.

scholarly journals Direct detection of circulating microRNA-122 using dynamic chemical labelling with single molecule detection overcomes stability and isomiR challenges for biomarker qualification

2019 ◽  
Author(s):  
Barbara López-Longarela ◽  
Emma E. Morrison ◽  
John D. Tranter ◽  
Lianne Chahman-Vos ◽  
Jean-François Léonard ◽  
...  
Keyword(s):  
Liver Injury ◽  
Single Molecule ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Circulating Microrna ◽  
Serum Samples ◽  
Drug Induced ◽  
Patients At Risk ◽  
Area Under Roc Curve ◽  
Fit For Purpose

AbstractCirculating microRNAs are biomarkers reported to be stable and translational across species. miR-122 (miR-122-5p) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). Our objective was to develop an extraction-free and amplification-free detection method for measuring miR-122 that has translational utility in context of DILI. We developed a single molecule dynamic chemical labelling (DCL) assay based on miR-122 hybridization to an abasic peptide nucleic acid probe that contained a reactive amine instead of a nucleotide at a specific position in the sequence. The single molecule DCL assay specifically measured miR-122 directly from 10 µL of serum or plasma without any extraction steps, with a fit-for-purpose limit of detection of 1.32 pM. In 192 human serum samples, DCL accurately identified patients at risk of DILI (area under ROC curve 0.98 (95%CI 0.96-1), P<0.0001). The miR-122 assay also quantified liver injury in rats and dogs. When DCL beads were added to serum, the miR-122 signal was stabilised (no loss of signal after 14 days at room temperature). By contrast, there was substantial degradation of miR-122 in the absence of beads (≈60% lost in 1 day). RNA sequencing demonstrated the presence of multiple miR-122 isomiRs with DILI that were at low concentration or not present in healthy patient serum. Sample degradation over time produced more isomiRs, particularly rapidly with DILI. PCR was inaccurate when analysing miR-122 isomiRs, whereas the DCL assay demonstrated accurate quantification. In summary, the DCL assay can accurately measure miR-122 directly from serum and plasma to diagnose liver injury in humans and other species, and can overcome important microRNA biomarker analytical and biological challenges.

scholarly journals Power-law behavior of transcription factor dynamics at the single-molecule level implies a continuum affinity model

2021 ◽  
Author(s):  
David A Garcia ◽  
Gregory Fettweis ◽  
Diego M Presman ◽  
Ville Paakinaho ◽  
Christopher Jarzynski ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Single Molecule ◽  
Power Law ◽  
Dwell Time ◽  
Specific Binding ◽  
Power Law Distribution ◽  
Single Molecule Level ◽  
Binding Behavior ◽  
Chromatin Template ◽  
Nuclear Domains

Abstract Single-molecule tracking (SMT) allows the study of transcription factor (TF) dynamics in the nucleus, giving important information regarding the diffusion and binding behavior of these proteins in the nuclear environment. Dwell time distributions obtained by SMT for most TFs appear to follow bi-exponential behavior. This has been ascribed to two discrete populations of TFs—one non-specifically bound to chromatin and another specifically bound to target sites, as implied by decades of biochemical studies. However, emerging studies suggest alternate models for dwell-time distributions, indicating the existence of more than two populations of TFs (multi-exponential distribution), or even the absence of discrete states altogether (power-law distribution). Here, we present an analytical pipeline to evaluate which model best explains SMT data. We find that a broad spectrum of TFs (including glucocorticoid receptor, oestrogen receptor, FOXA1, CTCF) follow a power-law distribution of dwell-times, blurring the temporal line between non-specific and specific binding, suggesting that productive binding may involve longer binding events than previously believed. From these observations, we propose a continuum of affinities model to explain TF dynamics, that is consistent with complex interactions of TFs with multiple nuclear domains as well as binding and searching on the chromatin template.

scholarly journals Deciphering the Disaggregation Mechanism of Amyloid Beta Aggregate by 4-(2-Hydroxyethyl)-1-Piperazinepropanesulfonic Acid Using Electrochemical Impedance Spectroscopy

Sensors ◽  
2021 ◽  
Vol 21 (3) ◽  
pp. 788
Author(s):  
Hien T. Ngoc Le ◽  
Sungbo Cho
Keyword(s):  
Electrochemical Impedance Spectroscopy ◽  
Impedance Spectroscopy ◽  
Electrochemical Impedance ◽  
Specific Binding ◽  
Amyloid Β ◽  
Electrochemical Measurement ◽  
Charge Transfer Resistance ◽  
Chemical Agent ◽  
Transfer Resistance ◽  
K Value

Aggregation of amyloid-β (aβ) peptides into toxic oligomers, fibrils, and plaques is central in the molecular pathogenesis of Alzheimer’s disease (AD) and is the primary focus of AD diagnostics. Disaggregation or elimination of toxic aβ aggregates in patients is important for delaying the progression of neurodegenerative disorders in AD. Recently, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) was introduced as a chemical agent that binds with toxic aβ aggregates and transforms them into monomers to reduce the negative effects of aβ aggregates in the brain. However, the mechanism of aβ disaggregation by EPPS has not yet been completely clarified. In this study, an electrochemical impedimetric immunosensor for aβ diagnostics was developed by immobilizing a specific anti-amyloid-β (aβ) antibody onto a self-assembled monolayer functionalized with a new interdigitated chain-shaped electrode (anti-aβ/SAM/ICE). To investigate the ability of EPPS in recognizing AD by extricating aβ aggregation, commercially available aβ aggregates (aβagg) were used. Electrochemical impedance spectroscopy was used to probe the changes in charge transfer resistance (Rct) of the immunosensor after the specific binding of biosensor with aβagg. The subsequent incubation of the aβagg complex with a specific concentration of EPPS at different time intervals divulged AD progression. The decline in the Rct of the immunosensor started at 10 min of EPPS incubation and continued to decrease gradually from 20 min, indicating that the accumulation of aβagg on the surface of the anti-aβ/SAM/ICE sensor has been extricated. Here, the kinetic disaggregation rate k value of aβagg was found to be 0.038. This innovative study using electrochemical measurement to investigate the mechanism of aβagg disaggregation by EPPS could provide a new perspective in monitoring the disaggregation periods of aβagg from oligomeric to monomeric form, and then support for the prediction and handling AD symptoms at different stages after treatment by a drug, EPPS.

scholarly journals Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alan M. Szalai ◽  
Bruno Siarry ◽  
Jerónimo Lukin ◽  
David J. Williamson ◽  
Nicolás Unsain ◽  
...  
Keyword(s):  
Single Molecule ◽  
Total Internal Reflection ◽  
Single Molecules ◽  
Fluorescence Microscope ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Axial Position ◽  
Localization Microscopy ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

AbstractSingle-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule’s image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

scholarly journals Analysis of anticoagulants for blood-based quantitation of amyloid β oligomers in the sFIDA assay

2017 ◽  
Vol 398 (4) ◽  
pp. 465-475 ◽  
Author(s):  
Kateryna Kravchenko ◽  
Andreas Kulawik ◽  
Maren Hülsemann ◽  
Katja Kühbach ◽  
Christian Zafiu ◽  
...  
Keyword(s):  
Blood Plasma ◽  
Limit Of Detection ◽  
Amyloid Β ◽  
Distribution Analysis ◽  
Aβ Oligomers ◽  
Quantitative Measurements ◽  
Suitable Parameter ◽  
Edta Plasma ◽  
Soluble Aβ Oligomers ◽  
Parameter Values

Abstract Early diagnostics at the preclinical stage of Alzheimer’s disease is of utmost importance for drug development in clinical trials and prognostic guidance. Since soluble Aβ oligomers are considered to play a crucial role in the disease pathogenesis, several methods aim to quantify Aβ oligomers in body fluids such as cerebrospinal fluid (CSF) and blood plasma. The highly specific and sensitive method surface-based fluorescence intensity distribution analysis (sFIDA) has successfully been established for oligomer quantitation in CSF samples. In our study, we explored the sFIDA method for quantitative measurements of synthetic Aβ particles in blood plasma. For this purpose, EDTA-, citrate- and heparin-treated blood plasma samples from five individual donors were spiked with Aβ coated silica nanoparticles (Aβ-SiNaPs) and were applied to the sFIDA assay. Based on the assay parameters linearity, coefficient of variation and limit of detection, we found that EDTA plasma yields the most suitable parameter values for quantitation of Aβ oligomers in sFIDA assay with a limit of detection of 16 fM.

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