A calibration-free approach to detecting microRNA with DNA-modified gold coated magnetic nanoparticles as dispersible electrodes

Gold coated magnetic nanoparticles (Au@MNPs), modified with DNA sequences give dispersible electrodes that can detect ultralow amounts of microRNAs and other nucleic acids but, as with most other sensors, they require calibration. Herein we show how to adapt a calibration free approach for electrochemical aptamer-based sensors on bulk electrodes to microRNA (miR-21) detection with methylene blue terminated DNA modified Au@MNPs. The electrochemical square wave voltammetry signal from the DNA-Au@MNPs when collected at a bulk electrode under magnetic control, decreases upon capture of miR-21. We show that the square wave voltammogram has concentration dependent and independent frequencies that can be used to give a calibration free signal

Biomolecular Binding under Confinement: Statistical Predictions of Steric Influence in Absence of Long-Distance Interactions

We propose a theoretical model for the influence of confinement on biomolecular binding at the single-molecule scale at equilibrium, based on the change of the number of microstates (localization and orientation) upon reaction. Three cases are discussed: DNA sequences shorter and longer than the single strain DNA Kuhn length and spherical proteins, confined into a spherical container (liposome, droplet, etc.). The influence of confinement is found to be highly dependent on the molecular structure and significant for large molecules (relative to container size)

Impact of the Coverage of Aptamers on a Nanoparticle on the Binding Equilibrium and Kinetics between Aptamer and Protein

Knowledge of the interaction between aptamer and protein is integral to the design and development of aptamer-based biosensors. Nanoparticles functionalized with aptamers are commonly used in these kinds of sensors. As such, studies into how the number of aptamers on the nanoparticle surface influence both kinetics and thermodynamics of the binding interaction are required. In this study, aptamers specific for interferon gamma (IFN-γ) were immobilized on the surface of gold nanoparticles (AuNPs), and the effect of surface coverage of aptamer on the binding interaction with its target was investigated using fluorescence spectroscopy. The number of aptamers were adjusted from an average of 9.6 to 258 per particle. The binding isotherm between AuNPs-aptamer conjugate and protein was modeled with the Hill-Langmuir equation, and the determined equilibrium dissociation constant (K′D) decreased 10-fold when increasing the coverage of aptamer. The kinetics of the reaction as a function of coverage of aptamer were also investigated, including the association rate constant (kon) and the dissociation rate constant (koff). The AuNPs-aptamer conjugate with 258 aptamers per particle had the highest kon, while the koff was similar for AuNPs-aptamer conjugates with different surface coverages. Therefore, the surface coverage of aptamers on AuNPs affects both the thermodynamics and the kinetics of the binding. The AuNPs-aptamer conjugate with the highest surface coverage is the most favorable in biosensors considering the limit of detection, sensitivity, and response time of the assay. These findings deepen our understanding of the interaction between aptamer and target protein on the particle surface, which is important to both improve the scientific design and increase the application of aptamer-nanoparticle based biosensor

Ultrasensitive detection of programmed death-ligand 1 (PD-L1) in whole blood using dispersible electrodes

The direct quantification of programmed death-ligand 1 (PD-L1) as a biomarker for cancer diagnosis, prognosis and treatment efficacy is an unmet clinical need. Herein, we demonstrate the first report of rapid, ultrasensitive and selective electrochemical detection of PD-L1directly in undiluted whole blood using modified gold-coated magnetic nanoparticles as “dispersible electrodes” with an ultralow detection limit of 15 attomolar and a response time of only 15 minutes

Single particle detection of protein molecules using dark-field microscopy to avoid signals from nonspecific adsorption

A massively parallel single particle sensing method based on core-satellite formation of Au nanoparticles was introduced for the detection of interleukin 6 (IL-6). This method exploits the fact that the localized plasmon resonance (LSPR) of the plasmonic nanoparticles will change as a result of core-satellite formation, resulting in a change in the observed color. In this method, the hue (color) value of thousands of 67 nm Au nanoparticles immobilized on a glass coverslip surface is analyzed by a Matlab code before and after the addition of reporter nanoparticles containing IL-6 as target protein. The average hue shift as the result of core-satellite formation is used as the basis to detect small amount of proteins. This method enjoys two major advantages. First it is able to analyze the hue values of thousands of nanoparticles in parallel in less than a minute. Secondly the method is able to circumvent the effect of non-specific adsorption, a major issue in the field of biosensing

Synthesis of gold-coated magnetic conglomerate nanoparticles with a fast magnetic response for bio-sensing

The versatile qualities of gold coated magnetic nanoparticles for both optical and electrochemical detection, as well as the separation of analytes, make them an excellent choice for ultrasensitive biosensing applications. The challenge with such nanoparticles however is that strongly magnetic nanoparticles that reach the magnet rapidly are prone to aggregation, whilst superparamagnetic nanoparticles that are stable against aggregation reach the magnet slowly. Here, we report a conglomerate nanostructure consisting of superparamagnetic nanoparticles coated with gold that provides a rapid magnetic response while exhibiting colloidal stability in solution. The performance of these gold coated magnetic nanoparticles for both bio-separation and biosensing was demonstrated for application in biosensors, dispersible electrodes for detecting microRNA and surface-enhanced Raman scattering

Locked nucleic acid molecular beacon for multiplex detection of loop mediated isothermal amplification

Loop mediated isothermal amplification (LAMP) holds incredible promise for point - of - care molecular diagnostics because of its high sensitivity and isothermal amplification behaviour. The issues related to the spurious non-specific amplification caused by the template independent amplification of primers itself causes false positive detection. This can be exacerbated by the common indirect methods used for detection of LAMP. Developing robust and specific detection methods for LAMP is a challenge due to the complex nature of the LAMP amplicons. To see wider adaptation of LAMP, we employ locked nucleic acid bases in molecular beacon to provide the structural stability to the hairpin probes that enable specific and multiplex detection of LAMP. Locked nucleic acid (LNA) modification provides ultra - high thermal stability to the molecular beacons resulting in negligible background fluorescence in the closed state. In this study, various combinations of LNA modifications in the stem and loop region were used and characterized for their thermal stability and influence on hybridization efficiency. The sequence specificity and ultra - high thermal stability of the LNA bases was exploited to develop a multiplex LAMP assay for detection of clinically important antibiotic resistance in S.aureus in 30 min. Multiplex approaches hold a significant advancement in LAMP and would find widespread applications in molecular diagnostics

DNA-hybridisation detection on Si(100) surfaces using lightactivated electrochemistry: a comparative study between bovine serum albumin and hexaethylene glycol as antifouling layers

Light can be used to spatially resolve electrochemical measurements on a semiconductor electrode. This phenomenon has been explored to detect DNA hybridization with light-addressable potentiometric sensors and, more recently, with light-addressable amperometric sensors based on organic-monolayer-protected Si(100). Here, a contribution to the field is presented by comparing sensing performances when bovine serum albumin (BSA) and hexaethylene glycol (OEG₆) are employed as antifouling layers that resist nonspecific adsorption to the DNA-modified interface on Si(100) devices. What is observed is that both sensors based on BSA or OEG₆ initially allow electrochemical distinction among complementary, noncomplementary, and mismatched DNA targets. However, only surfaces based on OEG₆ can sustain electroactivity over time. Our results suggest that this relates to accelerated SiO_<i>x</i> formation occasioned by BSA proteins adsorbing on monolayer-protected Si(100) surfaces. Therefore, DNA biosensors were analytically explored on low-doped Si(100) electrodes modified on the molecular level with OEG₆ as an antifouling layer. First, light-activated electrochemical responses were recorded over a range of complementary DNA target concentrations. A linear semilog relation was obtained from 1.0 × 10^–11 to 1.0 × 10^–6 mol L^–1 with a correlation coefficient of 0.942. Then, measurements with three independent surfaces indicated a relative standard deviation of 4.5%. Finally, selectivity tests were successfully performed in complex samples consisting of a cocktail mixture of four different DNA sequences. Together, these results indicate that reliable and stable light-activated amperometric DNA sensors can be achieved on Si(100) by employing OEG₆ as an antifouling layer