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.

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 An Electrochemical Strategy for the Simultaneous Detection of Doxorubicin and Simvastatin for Their Potential Use in the Treatment of Cancer

Biosensors ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 15
Author(s):  
Iulia Rus ◽  
Mihaela Tertiș ◽  
Cristina Barbălată ◽  
Alina Porfire ◽  
Ioan Tomuță ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Electrolyte Solution ◽  
Electrode Material ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Simultaneous Detection ◽  
Linear Sweep Voltammetry ◽  
Pharmaceutical Formulations ◽  
Promising Tool ◽  
Scan Rate

The aim of this study was to develop a disposable, simple, fast, and sensitive sensor for the simultaneous electrochemical detection of doxorubicin (DOX) and simvastatin (SMV), which could be used in preclinical studies for the development of new pharmaceutical formulations for drug delivery. Firstly, the electrochemical behavior of each molecule was analyzed regarding the influence of electrode material, electrolyte solution, and scan rate. After this, the proper electrode material, electrolyte solution, and scan rate for both active substances were chosen, and a linear sweep voltammetry procedure was optimized for simultaneous detection. Two chronoamperometry procedures were tested, one for the detection of DOX in the presence of SMV, and the other one for the detection of DOX and SMV together. Finally, calibration curves for DOX and SMV in the presence of each other were obtained using both electrochemical methods and the results were compared. The use of amperometry allowed for a better limit of detection (DOX: 0.1 μg/mL; SMV: 0.7 μg/mL) than the one obtained in voltammetry (1.5 μg/mL for both drugs). The limits of quantification using amperometry were 0.5 μg/mL for DOX (dynamic range: 0.5–65 μg/mL) and 2 μg/mL for SMV (dynamic range: 2–65 μg/mL), while using voltammetry 1 μg/mL was obtained for DOX (dynamic range: 1–100 μg/mL) and 5 μg/mL for SMV (dynamic range: 5–100 μg/mL). This detection strategy represents a promising tool for the analysis of new pharmaceutical formulations for targeted drug delivery containing both drugs, whose association was proven to bring benefits in the treatment of cancer.

scholarly journals A novel online coupling of ion selective electrode with the flow injection system for the determination of vitamin B1

2016 ◽  
Vol 13 (2) ◽  
pp. 458-469
Author(s):  
Baghdad Science Journal
Keyword(s):  
Flow Injection ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Vitamin B1 ◽  
Pharmaceutical Formulations ◽  
Ion Selective Electrode ◽  
Injection System ◽  
Selective Electrode ◽  
Flow Injection System

A simple, fast, selective of a new flow injection analysis method coupled with potentiometric detection was used to determine vitamin B1 in pharmaceutical formulations via the prepared new selective membranes. Two electrodes were constructed for the determination of vitamin B1 based on the ion-pair vitamin B1-phosphotungestic acid (B1-PTA) in a poly (vinyl chloride) supported with a plasticized di-butyl phthalate (DBPH) and di-butyl phosphate (DBP). Applications of these ion selective electrodes for the determination of vitamin B1 in the pharmaceutical preparations for batch and flow injection systems were described. The ion selective membrane exhibited a near-Nernstian slope values 56.88 and 58.53 mV / decade, with the linear dynamic range of vitamin B1 was 5 x 10-5- 1 x 10-2 and 1 x 10-4-1 x 10-2 mol.L-1, in batch and FIA, respectively. The limit of detection was 3.5 x 10-5 and 9.5 x 10-5 mol.L-1, with the percentage linearity 98.85 and 95.22 in batch and FIA, respectively. The suggested ion selective electrode has been utilized perfection in the determination of vitamin B1 in pharmaceutical formulations using batch and flow injection system, respectively.

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 Simultaneous Determination of Trace Mefenamic Acid in Pharmaceutical Samples via Flow Injection Fluorometry

2020 ◽  
Vol 10 (03) ◽  
pp. 395-401
Author(s):  
Mohammad K. Hammood ◽  
Maryam Hamed
Keyword(s):  
Flow Injection ◽  
Mefenamic Acid ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Basic Medium ◽  
Minimum Concentration ◽  
Pharmaceutical Samples ◽  
Fluorescence Measurements ◽  
532 Nm

Mefenamic acid belongs to non-steroidal anti-inflammatory drugs that are used widely for the treatment of analgesia. Our aim from this study is to establish a new assay for the quantitative determination of mefenamic acid (MFA) in the pharmaceutical sample by two sensitive and rapid flow injection-fluorometric methods. A homemade fluorometer was used in fluorescence measurements, which using solid-state laser diode 405 and 532 nm as a source, combined with a continuous flow injection technique. The first method depends on the effect of MFA on calcein blue (CLB) fluorescence at 405 nm. Another method is a study of rhodamine-6G (Rh-6G) fluorescence after adding MFA, and recording at 532 nm. Optimum parameters as fluorescent dye concentration, basic medium, flow rate, sample volume, purge time, and delay coil have been investigated. The dynamic range of MFA was 0.2 to 2 mmol.L-1; 0.5 to 2.3 mmol.L-1 with linearity percentage (% r2) 98.92 and 99.83%, for Rh-6G and CLB, respectively. Limit of detection at a minimum concentration in calibration curve 189.34 and 199.89 ng/sample, for Rh-6G and CLB, respectively. The comparison of developed methods with the classical method (UV-vis spectrophotometry) was achieved. The proposed methods were successfully applied for the determination of MFA in the pharmaceutical samples and can be used as an alternative method.

Gold nanoparticle-dotted, ionic liquid-functionalised, carbon hybrid material for ultra-sensitive detection of bisphenol A

2017 ◽  
Vol 14 (6) ◽  
pp. 385
Author(s):  
Yu Tian ◽  
Jianbo Li ◽  
Yanhui Wang ◽  
Chaofan Ding ◽  
Yuanling Sun ◽  
...  
Keyword(s):  
Ionic Liquid ◽  
Carboxylic Acid ◽  
Bisphenol A ◽  
Electrochemical Sensor ◽  
Water Solubility ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Hybrid Nanocomposites ◽  
Sensitive Detection ◽  
Endocrine Disorders

Environmental contextBisphenol A, an important industrial material widely used as a plasticiser, fire retardant and resin polymer material, can cause endocrine disorders and precocious puberty. We developed a portable and efficient method for determining bisphenol A, and apply it to the detection of bisphenol A in bottles for infants and young children. AbstractA highly effective electrochemical sensor was developed for the highly sensitive detection of bisphenol A (BPA). The sensor is based on a glassy carbon electrode modified with a composite comprising 1-butyl-3-methyl imidazole hydrobromide (an ionic liquid, IL)-functionalised grapheme oxide (GO) to which gold nanoparticles (AuNPs) and carboxylic acid-functionalised carbon nanotubes (CNT) were absorbed. The negatively charged carboxylic acid-functionalised CNTs and AuNPs are adsorbed on the positively charged GO-IL composite film by electrostatic adsorption. The as-prepared GO-IL-CNT-AuNP hybrid nanocomposites exhibit excellent water solubility owing to the high hydrophilicity of the GO-IL components. Moreover, the excellent conductivity is attributed to the good conductivity of the IL, CNT and AuNP components. The hydrid materials enhance the preconcentration efficiency of BPA and accelerate the electron transfer rate at the electrode–electrolyte interface, as such the resultant fabricated electrochemical sensor displays a fast, stable and sensitive detection performance for trace amounts of BPA. Differential pulse voltammetry was used as a sensitive analytical method for the determination of BPA, and a much wider linear dynamic range of BPA determination was found between 5 and 100nM. The limit of detection for BPA was found down to 1.5nM based on a signal to nose ratio of 3. The modified electrode was successfully employed to detect BPA extracted from a plastic water bottle and milk carton.

scholarly journals Highly sensitive dual mode electrochemical platform for microRNA detection

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Pawan Jolly ◽  
Marina R. Batistuti ◽  
Anna Miodek ◽  
Pavel Zhurauski ◽  
Marcelo Mulato ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Dynamic Range ◽  
Electrochemical Impedance ◽  
Limit Of Detection ◽  
Dual Mode ◽  
Electrode Surfaces ◽  
Microrna Detection ◽  
Highly Sensitive ◽  
Detection Mode ◽  
Amplification Strategy

Abstract MicroRNAs (miRNAs) play crucial regulatory roles in various human diseases including cancer, making them promising biomarkers. However, given the low levels of miRNAs present in blood, their use as cancer biomarkers requires the development of simple and effective analytical methods. Herein, we report the development of a highly sensitive dual mode electrochemical platform for the detection of microRNAs. The platform was developed using peptide nucleic acids as probes on gold electrode surfaces to capture target miRNAs. A simple amplification strategy using gold nanoparticles has been employed exploiting the inherent charges of the nucleic acids. Electrochemical impedance spectroscopy was used to monitor the changes in capacitance upon any binding event, without the need for any redox markers. By using thiolated ferrocene, a complementary detection mode on the same sensor was developed where the increasing peaks of ferrocene were recorded using square wave voltammetry with increasing miRNA concentration. This dual-mode approach allows detection of miRNA with a limit of detection of 0.37 fM and a wide dynamic range from 1 fM to 100 nM along with clear distinction from mismatched target miRNA sequences. The electrochemical platform developed can be easily expanded to other miRNA/DNA detection along with the development of microarray platforms.

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