== Magnetic nanotag-based protein detection assay

== Magnetic nanotag-based protein detection assay. in both research and clinical settings. Keywords:giant magnetoresistive sensors, GMR, magnetic JUN nanotags, multiplex protein detection, biochip A consensus is emerging that early detection and personalized treatment in clinics based on genetic and proteomic profiles of perhaps 420 biomarkers are the key to improving the survival rate of patients with complex diseases, such as cancer, autoimmune disorders, infectious diseases, and cardiovascular diseases (13). Although the tools for large-scale biomarker discovery with hundreds to thousands of biomarkers are available, there are few biomolecular detection tools capable of multiplex and sensitive detection of protein biomarkers that can be readily adopted in clinical settings for biomarker validation and for personalized diagnosis and treatment. This need, we believe, can be fulfilled by the magnetic nanotag (MNT)-based biomolecular assay technology reported here. We demonstrate the feasibility and implementation of this technology with multiple potential cancer markers. Several research groups are investigating MNT (46)-based analyte quantification as a highly sensitive alternative to optical biosensors and biochips (710). By labeling the target analyte of interest with MNTs (seeFig. 1), analyte detection and quantification can occur when the analyte binds to capture probes on the surface of giant magnetoresistive (GMR) sensors (1115) such as spin valve (SV) sensors (16), which have been developed and optimized for use in hard disk drives on a scale of hundreds of millions of units annually with great economy and reliability. Such sensors, when modified for use in biological applications, were previously shown to be capable of detecting as few as 10 MNTs (13,16). == Fig. 1. == Arterolane Magnetic nanotag-based protein detection assay. Probe sensors are functionalized with capture antibodies specific to the chosen analyte, whereas control sensors are blocked with a 1 wt% BSA solution (A). During analyte incubation, the probe sensors capture a fraction of the analyte molecules (B). A biotinylated linker antibody is subsequently incubated, which binds to the captured analyte (C), and which provides binding sites for the streptavidin-coated magnetic nanotags. Streptavidin-coated magnetic nanotags are then incubated (D), and the nanotag binding signal, which saturates Arterolane at an analyte concentration-dependent level, is used to quantify the analyte concentration. == Results == Given the recent efforts to develop methods for early cancer detection via quantification of cancer-related cytokines, we chose the following analytes for our MNT-based protein assays: cancer embryonic antigen (CEA), eotaxin, granulocyte colony-stimulating factor (G-CSF), interleukin-1-alpha (IL-1), interleukin-10 (IL-10), IFN gamma (IFN-), lactoferrin, and tumor necrosis factor alpha (TNF-).Fig. 1outlines the detection scheme Arterolane in which analyte is captured on the sensor surface and quantified with streptavidin-coated MNTs. Typically, the analyte and linker antibody incubation time (Fig. 1BandC) is 1 h or less, whereas the MNT-based quantification (Fig. 1D) requires <15 min. It is possible to reduce the total assay time to <30 min, for example through analyte incubation in a microfluidic channel (17), so that it is suitable for physician office laboratory or point of care applications. We currently implement the MNT detection scheme on a custom fabricated chip, which has an array of 64 SV sensors and a 200-l reaction well placed on top [seeSupporting Information (SI)], so that the reagents can be pipetted and aspirated easily. At present, our prototype electronic instrumentation (see methods) is able to record up to 64 sensors in real-time, with an update rate of 64 data points every 5 s. Some of the sensors are covered with epoxy and used to record an electronic reference signal, so that nonbiological signal components such as temperature drift can be corrected. Typical binding curve data (signal vs. time) from MNT-based immunoassays with multiple probes is shown inFig. 2. Arterolane The moment of MNT application is defined as t = 0. Beginning at t = 0, the signal rise reflects the binding of MNTs to the SV sensor surface in real time. As a result of the indirect labeling method used here, the real-time data contains information about the MNT-binding kinetics rather than the analyte binding kinetics, but the saturation level of an MNT-binding curve is taken as a direct Arterolane measure of binding site abundance on the sensor surface, which in turn is determined by the concentration of the previously applied analyte. In.