Antibody-decorated Nanoparticles Research Articles

Boosting the sensitivity of paper-based biosensors with polymeric water-soluble reservoirs

Storing highly concentrated reagents and releasing them with high efficiency is crucial for developing highly sensitive biosensors. However, the performance of reservoirs in biosensors made of filter paper is limited by the intrinsic physicochemical properties of untreated cellulose. In this article, the impact of 9 different polymer modifications on the storage and release of enzymes and nanoparticles in paper-based biosensors is studied for the first time. Carboxymethylcellulose, polyvinyl alcohol, carrageenan, and chitosan, which contain hydroxyl groups, yielded paper-based reservoirs with the highest concentration of reagents in them. Polymer reservoirs made of carboxymethylcellulose release enzymes rapidly and at high concentration, which results in colorimetric glucose biosensors with an ultralow limit of detection of 0.005 mM. Reservoirs made with a blend of polymers concentrate antibody-decorated nanoparticles and decrease 10 times the limit of detection of a model immunosensor. An adaption of this design was used for detecting the pathogen Klebsiella pneumoniae in urine samples from hospital patients at the infectious dose threshold rapidly and with high specificity. The fabrication of the reservoirs only requires drop-casting a polymer solution on the paper and drying. This procedure is compatible with the fabrication of origami biosensors, which are made from a single piece of filter paper.

Paper biosensors for detecting elevated IL-6 levels in blood and respiratory samples from COVID-19 patients.

Decentralizing COVID-19 care reduces contagions and affords a better use of hospital resources. We introduce biosensors aimed at detecting severe cases of COVID-19 in decentralized healthcare settings. They consist of a paper immunosensor interfaced with a smartphone. The immunosensors have been designed to generate intense colorimetric signals when the sample contains ultralow concentrations of IL-6, which has been proposed as a prognosis biomarker of COVID-19. This is achieved by combining a paper-based signal amplification mechanism with polymer-filled reservoirs for dispensing antibody-decorated nanoparticles and a bespoken app for color quantification. With this design we achieved a low limit of detection (LOD) of 10-3 pg mL-1 and semi-quantitative measurements in a wide dynamic range between 10-3 and 102 pg mL-1 in PBS. The assay time is under 10 min. The low LOD allowed us to dilute blood samples and detect IL-6 with an LOD of 1.3 pg mL-1 and a dynamic range up to 102 pg mL-1. Following this protocol, we were able to stratify COVID-19 patients according to different blood levels of IL-6. We also report on the detection of IL-6 in respiratory samples (bronchial aspirate, BAS) from COVID-19 patients. The test could be easily adapted to detect other cytokines such as TNF-α and IL-8 by changing the antibodies decorating the nanoparticles accordingly. The ability of detecting cytokines in blood and respiratory samples paves the way for monitoring local inflammation in the lungs as well as systemic inflammation levels in the body.

Doxorubicin/cisplatin co-loaded hyaluronic acid/chitosan-based nanoparticles for in vitro synergistic combination chemotherapy of breast cancer

Combination chemotherapy has attracted more and more attention in the field of anticancer treatment. Herein, a synergetic targeted combination chemotherapy of doxorubicin (DOX) and cisplatin in breast cancer was realized by HER2 antibody-decorated nanoparticles assembled from aldehyde hyaluronic acid (AHA) and hydroxyethyl chitosan (HECS). Cisplatin and DOX were successively conjugated onto AHA through chelation and Schiff’s base reaction, respectively, forming DOX/cisplatin-loaded AHA inner core. The core was sequentially complexed with HECS and targeting HER2 antibody-conjugated AHA. The formed near-spherical nanoplatform had an average size of ∼160 nm and a zeta potential of −28 mV and displayed pH-responsive surface charge reversal and drug release behaviors. HER2 receptor-mediated active targeting significantly enhanced the cellular uptake of nanoplatform. Importantly, DOX and cisplatin exhibited a synergistic cell-killing effect in human breast cancer MCF-7 cells. These results clearly indicate that the novel nanoplatform is promising for synergistic combination chemotherapy of breast cancer.

Restricted Proteolysis and LC-MS/MS To Evaluate the Orientation of Surface-Immobilized Antibodies.

The molecular orientation of antibodies immobilized on solid surfaces plays a significant role in the sensitivity of immunoassays and efficiency of protein isolation using antibody-decorated nanoparticles. Optimally, nearly all antibody binding sites should be available to bind. Here we report for the first time an LC-MS/MS approach to probe antibody orientation directly, utilizing sterically restricted proteolysis. Trypsin-decorated magnetic beads (MBs, 1.5 μm) were much larger than average antibody-free areas (55 × 55 nm) of oriented antibodies on MBs, restricting proteolysis to mainly Fab regions. Randomly attached antibodies on MB surfaces served as controls. The tryptic-hydrolyzed peptides were quantified using LC-MS/MS peptide analysis as markers for average positions of Fc and Fab of antibodies on the beads. Different patterns of digestion rates were found due to proteolysis of the oriented and nonoriented antibodies on MBs. For oriented antibodies, the peptides from outer Fab regions gave a much higher digestion rate than those from Fc regions, while for randomly immobilized antibodies digestion rates for Fab and Fc peptides were similar. This novel approach is a useful and convenient tool to characterize antibody orientation for immunoassays and other applications. The relative degree of orientation can be assessed using a metric Ro denoting amount of Fab marker peptides found divided by Fc + Fab marker peptides × 100%. Oriented antibodies on the MBs also provided more efficient antigen capture compared to randomly immobilized antibodies.

Resistive-Pulse Measurements with Nanopipettes: Detection of Vascular Endothelial Growth Factor C (VEGF-C) Using Antibody-Decorated Nanoparticles.

Quartz nanopipettes have recently been employed for resistive-pulse sensing of Au nanoparticles (AuNP) and nanoparticles with bound antibodies. The analytical signal in such experiments is the change in ionic current caused by the nanoparticle translocation through the pipette orifice. This paper describes resistive-pulse detection of cancer biomarker (Vascular Endothelial Growth Factor-C, VEGF-C) through the use of antibody-modified AuNPs and nanopipettes. The main challenge was to differentiate between AuNPs with attached antibodies for VEGF-C and antigen-conjugated particles. The zeta-potentials of these types of particles are not very different, and, therefore, carefully chosen pipettes with well-characterized geometry were necessary for selective detection of VEGF-C.

Minimizing the non-specific binding of nanoparticles to the brain enables active targeting of Fn14-positive glioblastoma cells

A major limitation in the treatment of glioblastoma (GBM), the most common and deadly primary brain cancer, is delivery of therapeutics to invading tumor cells outside of the area that is safe for surgical removal. A promising way to target invading GBM cells is via drug-loaded nanoparticles that bind to fibroblast growth factor-inducible 14 (Fn14), thereby potentially improving efficacy and reducing toxicity. However, achieving broad particle distribution and nanoparticle targeting within the brain remains a significant challenge due to the adhesive extracellular matrix (ECM) and clearance mechanisms in the brain. In this work, we developed Fn14 monoclonal antibody-decorated nanoparticles that can efficiently penetrate brain tissue. We show these Fn14-targeted brain tissue penetrating nanoparticles are able to (i) selectively bind to recombinant Fn14 but not brain ECM proteins, (ii) associate with and be internalized by Fn14-positive GBM cells, and (iii) diffuse within brain tissue in a manner similar to non-targeted brain penetrating nanoparticles. In addition, when administered intracranially, Fn14-targeted nanoparticles showed improved tumor cell co-localization in mice bearing human GBM xenografts compared to non-targeted nanoparticles. Minimizing non-specific binding of targeted nanoparticles in the brain may greatly improve the access of particulate delivery systems to remote brain tumor cells and other brain targets.

Detection and Identification of Virus Particles on a Microfluidic Platform

We have developed a nanoparticle-based assay to capture and detect targeted virus particles on a microfluidic platform. Antibody-decorated magnetic nanoparticles were used to capture virions and detection was afforded by measuring the change in magnetic birefringence relaxation rates. Vesicular Stomatitis Virus (VSV), a bullet-shaped, negative-strand RNA virus, is used as a model for air, water or blood-borne pathogenic viruses. Antibodies against the single envelope glycoprotein of either the Indiana or New Jersey strains of VSV were conjugated to 30nm superparamagnetic nanoparticles to generate the capturing reagent. The size distribution of the antibody-nanoparticle conjugates was determined using a CONTIN analysis of dynamic light scattering (DLS) data. Our results were confirmed by TEM analysis. Antibody-decorated nanoparticles were combined with one of the VSV strains and subjected to Ismagilov mixing, utilizing two immiscible phases in micro-channels to increase the speed of capture. Virus binding was determined by changes in hydrodynamic volume of the virus-nanoparticle complexes as measured by their birefringence relaxation rate under stopped flow conditions. In addition, the dynamics of particle complex formation and aggregation was studied by DLS at different scattering angles. We compared the results of these two methods for measuring time-dependent increases in the size of the nanoparticle/virus complexes. The birefringence relaxation method is more adaptable to field and other applications than DLS since it is relatively insensitive to particle concentration or to the presence of dust and sample impurities. Our approach is superior to existing techniques such as qPCR or ELISA assays due to the speed of detection and insensitivity to environmental contaminants. We are looking into the suitability of this assay for point-of-care applications.This work was supported in part by NIH grant R44 ES012515.