Multiplex protein assays based on real-time magnetic nanotag sensing

Abstract

Magnetic nanotags (MNTs) are a promising alternative to fluorescent labels in biomolecular detection assays, because minute quantities of MNTs can be detected with inexpensive giant magnetoresistive (GMR) sensors, such as spin valve (SV) sensors. However, translating this promise into easy to use and multilplexed protein assays, which are highly sought after in molecular diagnostics such as cancer diagnosis and treatment monitoring, has been challenging. Here, we demonstrate multiplex protein detection of potential cancer markers at subpicomolar concentration levels and with a dynamic range of more than four decades. With the addition of nanotag amplification, the analytic sensitivity extends into the low fM concentration range. The multianalyte ability, sensitivity, scalability, and ease of use of the MNT-based protein assay technology make it a strong contender for versatile and portable molecular diagnostics in both research and clinical settings.A consensus is emerging that early detection and personalized treatment in clinics based on genetic and proteomic profiles of perhaps 4-20 biomarkers are the key to improving the survival rate of patients with complex diseases, such as cancer, autoimmune disorders, infectious diseases, and cardiovascular diseases (1-3). 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 (4-6)-based analyte quantification as a highly sensitive alternative to optical biosensors and biochips (7-10). By labeling the target analyte of interest with MNTs (see Fig. 1), analyte detection and quantification can occur when the analyte binds to capture probes on the surface of giant magnetoresistive (GMR) sensors (11-15) 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). ResultsGiven 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 colonystimulating factor (G-CSF), interleukin-1-alpha (IL-1␣), interleukin-10 (IL-10), IFN gamma (IFN-␥), lactoferrin, and tumor necrosis factor alpha (TNF-␣). Fig. 1 outlines the detection scheme in which analyte is captured on the sensor surface and qu...

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