Co-detection: Ultra-reliable Nanoparticle-Based Electrical Detection of Biomolecules in the Presence of Large Background Interference

Contrary to popular belief, we report that background interference in conductimetric biochips can be exploited using a novel "co-detection" principle to significantly improve the reliability of detecting trace quantities of biomolecules. The technique called "co-detection" exploits the non-linear redundancy amongst synthetically patterned biomolecular logic circuits for deciphering the presence or absence of target biomolecules in a sample. In this paper, we demonstrate the "co-detection" principle on gold-nanoparticle based conductimetric soft-logic circuits which uses a silver-enhancement technique for signal amplification. Using co-detection, we have been able to measure a 1000 times improvement in the reliability of detecting mouse IgG at concentration levels that are 10^5^ lower than the concentration of rabbit IgG which serves as background interference

Signal amplification for thrombin impedimetric aptasensor : sandwich protocol and use of gold-streptavidin nanoparticles

In this work, we report a highly specific amplification strategy demonstrated for the ultrasensitive biosensing of thrombin with the use of gold-streptavidin nanoparticles (strep-AuNPs) and silver reduction enhancement. The biotinylated aptamer of thrombin was immobilized onto an avidin-graphite epoxy composite (AvGEC) electrode surface by affinity interaction between biotin and avidin; electrochemical impedance measurements were performed in a solution containing the redox marker ferrocyanide/ferricyanide. The change in interfacial charge transfer resistance (Rct) experimented by the redox marker, was recorded to confirm aptamer complex formation with target protein, thrombin (Thr), in a label-free first stage. A biotinylated second thrombin aptamer, with complementary recognition properties was then used in a sandwich approach. The addition of strep-AuNPs and silver enhancement treatment led to a further increment of Rct thus obtaining significant signal amplification. The AptThrBio1-Thr-AptThrBio2 sandwich formation was inspected by confocal microcopy after incubation with streptavidin quantum dots. In order to visualize the presence of gold nanoparticles, the same silver enhancement treatment was applied to electrodes already modified with the nanoparticle-sandwich conjugate, allowing direct observation by scanning electron microscopy (SEM). Results showed high sensitivity and selectivity for thrombin detection, with an improvement from ca. 4.7 pM in a simple assay to 0.3 pM in the amplified reported scheme

Development of Optical Biosensor Technologies for Cardiac Troponin Recognition

Acute myocardial infarction (AMI) is the leading cause of death among cardiovascular diseases. Among the numerous attempts to develop coronary marker concepts into clinical strategies, cardiac troponin is known as a specific marker for coronary events. The cardiac troponin concentration level in blood has been shown to rise rapidly for 4–10 days after onset of AMI, making it an attractive approach for a long diagnosis window for detection. The extremely low clinical sensing range of cardiac troponin levels consequently makes the methods of detection highly sensitive. In this review, by taking into consideration optical methods applied for cardiac troponin detection, we discuss the most commonly used methods of optical immunosensing and provide an overview of the various diagnostic cardiac troponin immunosensors that have been employed for determination of cardiac troponin over the last several years

Silver enhancement of Nanogold particles during freeze substitution for electron microscopy

Recent advances in rapid freezing and fixation by freeze substitution have allowed structural cell biologists to apply these reliable modes of sample preparation to a wide range of specimens and scientific problems. Progress in electron tomography has produced cellular images with resolution approaching 4 nm in 3D, but our ability to localize macromolecules in these well-fixed, well-resolved samples has remained limited. When light fixation and low temperature embedding are employed with appropriate resins, immuno-localizations can recognize antigens at a section's surface, but labelling is therefore confined, not throughout the section's depth. Small, electron-dense markers, like Nanogold®, will often enter a living cell, serving as reliable tracers for endocytic activity, but these markers are usually too small to be visible in the context of a cell. We have developed a method for the silver enhancement of Nanogold particles that works during freeze substitution in organic solvents at low temperature. Here, we describe the development of this method, based on in vitro tests of reagents and conditions. We then show results from application of the method to an in vivo system, using Nanogold to track the internalization of immunoglobulin by neonatal murine intestinal epithelium, a specific example of receptor-mediated membrane traffic

Mechanism of active targeting in solid tumors with transferrin-containing gold nanoparticles

PEGylated gold nanoparticles are decorated with various amounts of human transferrin (Tf) to give a series of Tf-targeted particles with near-constant size and electrokinetic potential. The effects of Tf content on nanoparticle tumor targeting were investigated in mice bearing s.c. Neuro2A tumors. Quantitative biodistributions of the nanoparticles 24 h after i.v. tail-vein injections show that the nanoparticle accumulations in the tumors and other organs are independent of Tf. However, the nanoparticle localizations within a particular organ are influenced by the Tf content. In tumor tissue, the content of targeting ligands significantly influences the number of nanoparticles localized within the cancer cells. In liver tissue, high Tf content leads to small amounts of the nanoparticles residing in hepatocytes, whereas most nanoparticles remain in nonparenchymal cells. These results suggest that targeted nanoparticles can provide greater intracellular delivery of therapeutic agents to the cancer cells within solid tumors than their nontargeted analogs

Nanodiamond landmarks for subcellular multimodal optical and electron imaging.

There is a growing need for biolabels that can be used in both optical and electron microscopies, are non-cytotoxic, and do not photobleach. Such biolabels could enable targeted nanoscale imaging of sub-cellular structures, and help to establish correlations between conjugation-delivered biomolecules and function. Here we demonstrate a sub-cellular multi-modal imaging methodology that enables localization of inert particulate probes, consisting of nanodiamonds having fluorescent nitrogen-vacancy centers. These are functionalized to target specific structures, and are observable by both optical and electron microscopies. Nanodiamonds targeted to the nuclear pore complex are rapidly localized in electron-microscopy diffraction mode to enable "zooming-in" to regions of interest for detailed structural investigations. Optical microscopies reveal nanodiamonds for in-vitro tracking or uptake-confirmation. The approach is general, works down to the single nanodiamond level, and can leverage the unique capabilities of nanodiamonds, such as biocompatibility, sensitive magnetometry, and gene and drug delivery

A digital microarray using interferometric detection of plasmonic nanorod labels

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and dynamic range of traditional fluorescence microarrays compared to other techniques have been the technology's Achilles' Heel, and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule ('digital') regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform's primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about three orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10x objective lens. This approach does not require any chemical enhancement such as silver deposition, and scans arrays with a throughput similar to commercial fluorescence devices. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about one million directly from a single scan