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).

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-L1 directly 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.

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.

Smartphone technology facilitates point-of-care nucleic acid diagnosis: a beginner's guide.

The reliable detection of nucleic acids at low concentrations in clinical samples like blood, urine and saliva, and in food can be achieved by nucleic acid amplification methods. Several portable and hand-held devices have been developed to translate these laboratory-based methods to point-of-care (POC) settings. POC diagnostic devices could potentially play an important role in environmental monitoring, health, and food safety. Use of a smartphone for nucleic acid testing has shown promising progress in endpoint as well as real-time analysis of various disease conditions. The emergence of smartphone-based POC devices together with paper-based sensors, microfluidic chips and digital droplet assays are used currently in many situations to provide quantitative detection of nucleic acid targets. State-of-the-art portable devices are commercially available and rapidly emerging smartphone-based POC devices that allow the performance of laboratory-quality colorimetric, fluorescent and electrochemical detection are described in this review. We present a comprehensive review of smartphone-based POC sensing applications, specifically on microbial diagnostics, assess their performance and propose recommendations for the future.

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.

Optical tweezers-based characterisation of gold core-satellite plasmonic nano-assemblies incorporating thermo-responsive polymers.

We report on the characterisation of the optical properties and dynamic behaviour of optically trapped single stimuli-responsive plasmonic nanoscale assemblies. Nano-assemblies consist of a core-satellite arrangement where the constituent nanoparticles are connected by the thermoresponsive polymer, poly(DEGA-co-OEGA). The optical tweezers allow the particles to be held isolated in solution and interrogated using dark-field spectroscopy. Additionally, controlling the optical trapping power provides localised heating for probing the thermal response of the nanostructures. Our results identify a number of distinct core-satellite configurations that can be stably trapped, which are verified using finite element modelling. Laser heating of the nanostructures through the trapping laser yields irreversible modification of the arrangement, as observed through the scattering spectrum. We consider which factors may be responsible for the observed behaviour in the context of the core-satellite geometry, polymer-solvent interaction, and the bonding of the nanoparticles.

The use of a personal glucose meter for detecting procalcitonin through glucose encapsulated within liposomes.

Herein, a glucose meter-based immunosensing platform is developed that allows the quantification of procalcitonin (PCT) in whole blood samples. PCT is a biomarker for sepsis and its early detection would improve the safety of the patient, as the diagnostic process will be easier and faster. The method employs liposomes with encapsulated glucose as a signal generation tag, which are then used in a sandwich immunoassay by conjugating an antibody to the liposome. The optimal liposomes' size and concentration of encapsulated glucose is determined experimentally to be 200 nm and 27.8 mM, respectively. Upon the addition of a surfactant (Triton X-100), the glucose is released and a signal is detected with a personal glucose meter (PGM). This signal is directly proportional to the concentration of the PCT in the sample. The dynamic range of the assay developed was 0.153-15.38 nM, and could allow the detection of PCT as low as 0.15 nM. The assay showed a high selectivity toward PCT against other proteins such as C-reactive protein and human serum albumin and good reproducibility. This assay was able to quantitatively determine the amount of PCT in whole blood samples at clinically-relevant concentrations.

A portable nucleic acid detection system using natural convection combined with a smartphone.

The development of portable nucleic acid diagnostic devices has the potential to expand the availability of molecular diagnostics into low-resource settings. One of the promising solutions for rapid and simple DNA amplification is the use of Rayleigh-Bernard natural convection which is caused by a buoyancy-driven thermal gradient of liquid when heated from below. This natural convection avoids the use of the complex and sophisticated hardware that is required for precise maintenance of temperature cycles in conventional PCR. We have developed a stand-alone convective PCR (cPCR) device linked to a smartphone for rapid detection of nucleic acids using natural convection heating. The device amplifies multiple DNA samples simultaneously using a custom-made heat block controlled by Bluetooth wireless communication. The entire device is highly portable, user-friendly, battery-operated and can provide target DNA amplification in less than 30 min. A detection limit of 2.8 × 103 copies of a segment of lambda DNA was obtained when the two different fluorescently-tagged amplicons were collected magnetically and detected using the smartphone fluorescence reader. Thus, the combination of cPCR and multiplex fluorescence-based detection on a smartphone provides new opportunities for the development of affordable and portable molecular diagnostic devices for point-of-care situations or remote clinical settings.

DNA-Hybridization Detection on Si(100) Surfaces Using Light-Activated 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 x 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.

Systematic review of the impact of point-of-care testing for influenza on the outcomes of patients with acute respiratory tract infection.

Acute respiratory tract infections are a major cause of morbidity and mortality and represent a significant burden on the health care system. Laboratory testing is required to definitively distinguish infecting influenza virus from other pathogens, resulting in prolonged emergency department (ED) visits and unnecessary antibiotic use. Recently available rapid point-of-care tests (POCT) may allow for appropriate use of antiviral and antibiotic treatments and decrease patient lengths of stay. We undertook a systematic review to assess the effect of POCT for influenza on three outcomes: (1) antiviral prescription, (2) antibiotic prescription, and (3) patient length of stay in the ED. The databases Medline and Embase were searched using MeSH terms and keywords for influenza, POCT, antivirals, antibiotics, and length of stay. Amongst 245 studies screened, 30 were included. The majority of papers reporting on antiviral prescription found that a positive POCT result significantly increased use of antivirals for influenza compared with negative POCT results and standard supportive care. A positive POCT result also led to decreased antibiotic use. The results of studies assessing the effect of POCT on ED length of stay were not definitive. The studies assessed in this systematic review support the use of POCT for diagnosis of influenza in patients suffering an acute respiratory infection. Diagnosis using POCT may lead to more appropriate prescription of treatments for infectious agents. Further studies are needed to assess the effect of POCT on the length of stay in ED.