An umbrella for the cytokine storm: Enabling precision detection of CRS in CAR-T therapy.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14223-e14223
Author(s):  
Qimin Quan
Keyword(s):  
Single Molecule ◽  
Biomarker Discovery ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Predictive Biomarkers ◽  
Small Sample ◽  
Detection Sensitivity ◽  
Patient Death ◽  
Wide Range ◽  
Car T

e14223 Background: Cytokine release syndrome (CRS), a systemic inflammatory response observed with monoclonal antibody drugs and adoptive T cell treatments, has become a major issue for CAR-T therapy. CRS can present as a mild reaction requiring minimally invasive supportive care up to a severe systemic response resulting in patient death. Monitoring this response during these therapeutic treatments is non-trivial due the wide range of biomarker concentrations, small sample volumes, and long assay times. Current analytical methods are unable to address these needs, limiting the precision of CAR-T therapy and effective management of its side effects. Methods: Emerging studies in this area have focused in establishing a panel of predictive biomarkers to manage dosing and early interventions, among them, IFNγ, IL6, TNFα, MIP1 have shown predicative powers in pediatric patients. Nevertheless, a significant improvement (100x) on the detection sensitivity is required to predict the CRS response with currently available methods. In addition, CRS-associated biomarkers including CRP and ferritin vary from 10ng/mL-10mg/mL while other predictive biomarkers (eg, IL6, IFNγ, etc.) vary from 1pg/mL-100ng/mL. At present, no analytical tool, known to us, can provide this large dynamic range ( > 9 logs), with the requisite lower limit of detection, in a rapid single test to predict and differentiate low, medium or high grade responses. Results: We present the NanoMosaic platform, the technology that has requisite sensitivity and breadth of dynamic range to enable precision detection based on precise quantitation of CRS-relevant biomarkers. NanoMosaic technology is enabled by single molecule nanoneedle sensors that are densely integrated on a silicon chip and manufactured with a CMOS-compatible process. Absolute quantitation is achieved by imaging the spectrum of nanoneedles, corresponding directly to the number of molecules. Conclusions: Direct comparison of different protein biomarkers with orders of magnitude concentration variations becomes possible in one platform and small sample sizes. We envision NanoMosaic technology will not only drive biomarker discovery, but also enable precise dosing management for CAR-T therapy.

scholarly journals Rapid single-molecule digital detection of protein biomarkers for continuous monitoring of systemic immune disorders

Blood ◽  
2020 ◽  
Author(s):  
Yujing Song ◽  
Erin Sandford ◽  
Yuzi Tian ◽  
Qingtian Yin ◽  
Andrew G. Kozminski ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Clinical Care ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Biomarkers ◽  
Car T ◽  
Rapid Monitoring ◽  
Robust Measurements ◽  
Protein Assays

Digital protein assays have great potential to advance immunodiagnostics because of their single-molecule sensitivity, high precision, and robust measurements. However, translating digital protein assays to acute clinical care has been challenging because it requires their deployment with a rapid turnaround. Herein, we present a technology platform for ultra-fast digital protein biomarker detection by employing single-molecule counting of immune-complex formation events at an early, pre-equilibrium state. This method, which we term "pre-equilibrium digital enzyme-linked immunosorbent assay" (PEdELISA), can quantify a multiplexed panel of protein biomarkers in 10 µL of serum within an unprecedented assay incubation time of 15-300 sec over a 104 dynamic range. PEdELISA allowed us to perform rapid monitoring of protein biomarkers in patients manifesting post-chimeric antigen receptor T-cell (CAR-T) therapy cytokine release syndrome (CRS), with ~30 min sample-to-answer time and a sub-pg/mL limit of detection (LOD). The rapid, sensitive, and low input volume biomarker quantification enabled by PEdELISA is broadly applicable to timely monitoring of acute disease, potentially enabling more personalized treatment.

An Integrated Platform for Single Molecule Free Solution Hydrodynamic Separation Using Yoctomoles of DNA and Picoliter Samples

2012 ◽  
Author(s):  
Kelvin J. Liu ◽  
Tushar D. Rane ◽  
Yi Zhang ◽  
Cyrus W. Beh ◽  
Sarah M. Friedrich ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dynamic Range ◽  
Separation Efficiency ◽  
Limit Of Detection ◽  
Detection Sensitivity ◽  
Free Solution ◽  
Quantitative Accuracy ◽  
Hydrodynamic Separation ◽  
Exclusion Mechanism ◽  
The Cost

We report a microfluidic platform for sizing and separation of DNA called PicoSep that is among the most sensitive to date, requiring only yoctomoles of DNA (10−24 moles) and picoliters of sample (<10 pL). The cohesive integration of cylindrical illumination confocal spectroscopy based (CICS) single molecule counting with free solution hydrodynamic separation increases detection sensitivity >103-fold over traditional laser induced fluorescence (LIF) and vastly improves quantification accuracy. Separation is performed via single molecule free solution hydrodynamic separation (SML-FSHS). SML-FSHS relies on the wall exclusion mechanism to separate DNA as it is driven down buffer filled microchannel. High separation efficiency (plate number = 105 – 106) and high sizing resolution (37 bp – 2.1 kbp) are obtained across a wide dynamic range of DNA (100 bp – 27 kbp) in a single separation. Quantitative accuracy up to the limits imposed by molecular shot noise is achieved, and a limit of detection <20 molecules is demonstrated. Through the development of PicoSep, we have significantly reduced the cost and complexity of single molecule instrumentation while achieving sizing and quantification performance that surpasses capillary electrophoresis (CE). Unlike CE, no viscous sieving matrices, high voltage power supplies, or capillary wall coatings are necessary, making devices inexpensive, simple to fabricate, and easy to incorporate into lab on a chip systems.

scholarly journals Application of biosynthesized metal nanoparticles in electrochemical sensors

2021 ◽  
pp. 77-77
Author(s):  
Totka Dodevska ◽  
Dobrin Hadzhiev ◽  
Ivan Shterev ◽  
Yanna Lazarova
Keyword(s):  
Green Synthesis ◽  
Metal Nanoparticles ◽  
Electrochemical Sensors ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Electrochemical Sensing ◽  
Synthesis Methods ◽  
Range Limit ◽  
Wide Range

Recently, the development of eco-friendly, cost-effective and reliable methods for synthesis of metal nanoparticles has drawn a considerable attention. The so-called green synthesis, using mild reaction conditions and natural resources as plant extracts and microorganisms, has established as a convenient, sustainable, cheap and environmentally safe approach for synthesis of a wide range of nanomaterials. Over the past decade, biosynthesis is regarded as an important tool for reducing the harmful effects of traditional nanoparticle synthesis methods commonly used in laboratories and industry. This review emphasizes the significance of biosynthesized metal nanoparticles in the field of electrochemical sensing. There is increasing evidence that green synthesis of nanoparticles provides a new direction in designing of cost-effective, highly sensitive and selective electrode-catalysts applicable in food, clinical and environmental analysis. The article is based on 157 references and provided a detailed overview on the main approaches for green synthesis of metal nanoparticles and their applications in designing of electrochemical sensor devices. Important operational characteristics including sensitivity, dynamic range, limit of detection, as well as data on stability and reproducibility of sensors have also been covered. Keywords: biosynthesis; green synthesis; nanomaterials; nanotechnology; modified electrodes

scholarly journals Re-evaluating the conventional wisdom about binding assays

2020 ◽  
Author(s):  
Brandon D. Wilson ◽  
H. Tom Soh
Keyword(s):  
Molecular Recognition ◽  
Conceptual Understanding ◽  
Single Molecule ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Conventional Wisdom ◽  
Binding Assays ◽  
Digital Measurements ◽  
Assay Design ◽  
Recognition Systems

AbstractAnalytical technologies based on binding assays have evolved substantially since their inception nearly 60 years ago, but our conceptual understanding of molecular recognition has not kept pace. Indeed, contemporary technologies such as single-molecule and digital measurements have challenged, or even rendered obsolete, core aspects of the conventional wisdom related to binding assay design. Here, we explore the fundamental principles underlying molecular recognition systems, which we consider in terms of signals generated through concentration-dependent shifts in equilibrium. We challenge certain orthodoxies related to binding-based detection assays, including the primary importance of a low KD and the extent to which this parameter constrains dynamic range and limit of detection. Lastly, we identify key principles for designing binding assays optimally suited for a given detection application.

scholarly journals High resolution biosensor to test the capping level and integrity of mRNAs

2020 ◽  
Vol 48 (22) ◽  
pp. e129-e129
Author(s):  
Ignacio Moya-Ramírez ◽  
Clement Bouton ◽  
Cleo Kontoravdi ◽  
Karen Polizzi
Keyword(s):  
Single Molecule ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Chimeric Protein ◽  
Single Step ◽  
Quality Analysis ◽  
Eukaryotic Mrnas ◽  
In Vitro Capping

Abstract 5′ Cap structures are ubiquitous on eukaryotic mRNAs, essential for post-transcriptional processing, translation initiation and stability. Here we describe a biosensor designed to detect the presence of cap structures on mRNAs that is also sensitive to mRNA degradation, so uncapped or degraded mRNAs can be detected in a single step. The biosensor is based on a chimeric protein that combines the recognition and transduction roles in a single molecule. The main feature of this sensor is its simplicity, enabling semi-quantitative analyses of capping levels with minimal instrumentation. The biosensor was demonstrated to detect the capping level on several in vitro transcribed mRNAs. Its sensitivity and dynamic range remained constant with RNAs ranging in size from 250 nt to approximately 2700 nt and the biosensor was able to detect variations in the capping level in increments of at least 20%, with a limit of detection of 2.4 pmol. Remarkably, it also can be applied to more complex analytes, such mRNA vaccines and mRNAs transcribed in vivo. This biosensor is an innovative example of a technology able to detect analytically challenging structures such as mRNA caps. It could find application in a variety of scenarios, from quality analysis of mRNA-based products such as vaccines to optimization of in vitro capping reactions.

From High Performance Protein Micro Chip Toward Ultra High Sensitive Single Molecule Nano Array

2009 ◽  
Author(s):  
Fan-Gang Tseng
Keyword(s):  
Single Molecule ◽  
High Performance ◽  
Dynamic Range ◽  
Early Stage ◽  
High Dynamic Range ◽  
Disease Diagnosis ◽  
Protein Microarrays ◽  
Detection Sensitivity ◽  
High Throughput Analysis ◽  
High Efficient

Protein microarrays have been employed to screen tens to thousands of proteins simultaneously for the observation of the biochemical activities in the protein-protein, protein-nucleic acid and small molecule interactions. This technology allows high throughput analysis and holds great potential for basic molecular biology research, disease marker identification, toxicological response profiling and pharmaceutical target screening. However, proteins easily malfunction in harsh environments so that they are hardly preserved before the application because of their complex and fragile structures. On the other hand, identify scarce amount of proteins less than fM range is very important and challenge for disease diagnosis at very early stage. As a result, the procedures for protein micro array formation are very important for preserving protein functionality to ensure useful protein assays, as well as the improvement of the detection sensitivity up to single molecule event but with high dynamic range for disease early detection. Therefore, this paper provides a novel view from the preparation of high efficient protein micro chip toward ultra high sensitive single protein nano array through the technology integration of BioMEMS and Bio-Nanotechnology.

scholarly journals A theoretical framework for proteome-scale single-molecule protein identification using multi-affinity protein binding reagents

2021 ◽  
Author(s):  
Jarrett D Egertson ◽  
Dan DiPasquo ◽  
Alana Killeen ◽  
Vadim Lobanov ◽  
Sujal Patel ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Identification ◽  
Dynamic Range ◽  
False Negative ◽  
Theoretical Framework ◽  
Human Proteome ◽  
Single Experiment ◽  
Affinity Reagents ◽  
Wide Range ◽  
Biological Insight

The proteome is perhaps the most dynamic and valuable source of functional biological insight. Current proteomic techniques are limited in their sensitivity and throughput. A typical single experiment measures no more than 8% of the human proteome from blood or 35% from cells and tissues. Here, we introduce a theoretical framework for a fundamentally different approach to proteomics that we call Protein Identification by Short-epitope Mapping (PrISM). PrISM utilizes multi-affinity reagents to target short linear epitopes with both a high affinity and low specificity. PrISM further employs a novel protein decoding algorithm that considers the stochasticity expected for single-molecule binding. In simulations, PrISM is able to identify more than 98% of proteins across the proteomes of a wide range of organisms. PrISM is robust to potential experimental confounders including false negative detection events and noise. Simulations of the approach with a chip containing 10 billion protein molecules show a dynamic range of 11.5 and 9.5 orders of magnitude for blood plasma and HeLa cells, respectively. If implemented experimentally, PrISM stands to rapidly quantify over 90% of the human proteome in a single experiment, potentially revolutionizing proteomics research.

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.

Ultrasensitive SERS-Based Immunoassay of Tumor Marker in Serum Using Au–Ag Alloy Nanoparticles and Ag/AgBr Hybrid Nanostructure

NANO ◽  
2018 ◽  
Vol 13 (01) ◽  
pp. 1850001 ◽  
Author(s):  
Yongfeng Gao ◽  
Yuanhui Feng ◽  
Lu Zhou ◽  
Lucia Petti ◽  
Zhe Wang ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Healthy Person ◽  
Detection Sensitivity ◽  
Hybrid Nanostructure ◽  
Serum Samples ◽  
Broad Dynamic Range ◽  
Surface Enhanced Raman ◽  
Relative Errors

Ultrasensitive detection of alpha-fetoprotein (AFP) is critical for the early diagnosis of liver cancer. In this work, a novel surface-enhanced Raman scattering (SERS)-based immunoassay complex has been successfully developed for the detection of AFP by using the Au-Ag alloy nanoparticals and the Ag/AgBr hybrid nanostructure. As the typical bimetal or metal/semiconductor plasmonic materials, besides the strong SERS enhancement characteristics, the Au-Ag alloy nanoparticals exhibit excellent monodispersity and the Ag/AgBr hybrid nanostructure demonstrates good stability. The experimental results show that the SERS-based immunoassay of AFP presents a low limit of detection of 1.86[Formula: see text]fg/mL and a broad dynamic range from 2[Formula: see text]fg/mL to 0.8[Formula: see text][Formula: see text]g/mL. Furthermore, the clinical applicability of the proposed SERS-based immunoassay has been assessed by the detection of AFP in the human serum samples of cancer patient and healthy person. The test data are consistent well with that of chemiluminescence immunoassay (CLIA) in the relative errors of [Formula: see text]8.82–8.06% and show better detection sensitivity. It reveals that the proposed immunoassay protocol is significant for giving insight into the design of ultrasensitive biosensor and the point-of-care testing of cancers.

scholarly journals Comparison of Metal Nanoparticles (Au, Ag, Eu, Cd) Used for Immunoanalysis Using LA-ICP-MS Detection

Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 630
Author(s):  
Marcela Vlcnovska ◽  
Aneta Stossova ◽  
Michaela Kuchynka ◽  
Veronika Dillingerova ◽  
Hana Polanska ◽  
...  
Keyword(s):  
Metal Nanoparticles ◽  
Inductively Coupled Plasma ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
P53 Protein ◽  
Metal Nanoparticle ◽  
Biogenic Elements ◽  
Dot Blot ◽  
Icp Ms ◽  
Wide Range

Immunochemical methods are used not only in clinical practice for the diagnosis of a wide range of diseases but also in basic and advanced research. Based on the unique reaction between the antibody and its respective antigens, it serves to specifically recognize target molecules in biological complex samples. Current methods of labelling antibodies with elemental labels followed by detection by inductively coupled plasma mass spectrometry (ICP-MS) allow detection of multiple antigens in parallel in a single analysis. Using the laser ablation (LA) modality (LA-ICP-MS), it is also possible to monitor the spatial distribution of biogenic elements. Moreover, the employment of metal nanoparticle-labeled antibodies expands the applicability also to molecular imaging by LA-ICP-MS. In this work, conjugates of model monoclonal antibody (DO-1, recognizing p53 protein) with various metal nanoparticles-based labels were created and utilized in dot-blot analysis in order to compare their benefits and disadvantages. Based on experiments with the p53 protein standard, commercial kits of gold nanoparticles proved to be the most suitable for the preparation of conjugates. The LA-ICP-MS demonstrated very good repeatability, wide linear dynamic range (0.1–14 ng), and limit of detection was calculated as a 1.3 pg of p53 protein.

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