Advances in biosensor technologies for in vitro diagnostics have the potential to transform the practice of medicine. Despite considerable work in the biosensor field, there is still no general sensing platform that can be ubiquitously applied to detect the constellation of biomolecules in diverse clinical samples (for example, serum, urine, cell lysates or saliva) with high sensitivity and large linear dynamic range. A major limitation confounding other technologies is signal distortion that occurs in various matrices due to heterogeneity in ionic strength, pH, temperature and autofluorescence. Here we present a magnetic nanosensor technology that is matrix insensitive yet still capable of rapid, multiplex protein detection with resolution down to attomolar concentrations and extensive linear dynamic range. The matrix insensitivity of our platform to various media demonstrates that our magnetic nanosensor technology can be directly applied to a variety of settings such as molecular biology, clinical diagnostics and biodefense.

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...

Monitoring the kinetics of protein interactions on a high density sensor array is vital to drug development and proteomic analysis. Label-free kinetic assays based on surface plasmon resonance are the current gold standard, but they have poor detection limits, suffer from non-specific binding, and are not amenable to high throughput analyses. Here we show that magnetically responsive nanosensors that have been scaled to over 100,000 sensors/cm2 can be used to measure the binding kinetics of various proteins with high spatial and temporal resolution. We present an analytical model that describes the binding of magnetically labeled antibodies to proteins that are immobilized on the sensor surface. This model is able to quantify the kinetics of antibody-antigen binding at sensitivities as low as 20 zeptomoles of solute.

The clinical use of metallic expandable intravascular stents has resulted in improved therapeutic outcomes for coronary artery disease. However, arterial reobstruction after stenting, in-stent restenosis, remains an important problem. Gene therapy to treat in-stent restenosis by using gene vector delivery from the metallic stent surfaces has never been demonstrated. The present studies investigated the hypothesis that metal-bisphosphonate binding can enable site-specific gene vector delivery from metal surfaces. Polyallylamine bisphosphonate (PAA-BP) was synthesized by using Michael addition methodology. Exposure to aqueous solutions of PAA-BP resulted in the formation of a monomolecular bisphosphonate layer on metal alloy surfaces (steel, nitinol, and cobaltchromium), as demonstrated by x-ray photoelectron spectroscopy. Surface-bound PAA-BP enabled adenoviral (Ad) tethering due to covalent thiol-binding of either anti-Ad antibody or a recombinant Ad-receptor protein, D1. In arterial smooth muscle cell cultures, alloy samples configured with surface-tethered Ad were demonstrated to achieve site-specific transduction with a reporter gene, (GFP). Rat carotid stent angioplasties using metal stents exposed to aqueous PAA-BP and derivatized with anti-knob antibody or D1 resulted in extensive localized Ad-GFP expression in the arterial wall. In a separate study with a model therapeutic vector, Adinducible nitric oxide synthase (iNOS) attached to the bisphosphonate-treated metal stent surface via D1, significant inhibition of restenosis was demonstrated (neointimal͞media ratio 1.68 ؎ 0.27 and 3.4 ؎ 0.35; Ad-iNOS vs. control, P < 0.01). It is concluded that effective gene vector delivery from metallic stent surfaces can be achieved by using this approach.gene therapy ͉ local delivery ͉ restenosis T he use of balloon expandable metallic stents has resulted in improved therapeutic outcomes for coronary artery disease (1). However, stent angioplasty is complicated in many patients by reobstruction due to the formation of a neointima in the stented arterial segment, a disease process known as in-stent restenosis (2). The mechanisms responsible for instent restenosis involve proliferation and migration of medial smooth muscle cells (SMCs) and an associated increase in extracellular matrix components (2). The use of polymercoated drug-eluting stents has markedly decreased the incidence of in-stent restenosis observed with unmodified metal stents (3). However, both experimental (4) and clinical (5) studies indicate a number of concerns about this approach, because polymer coatings on stents cause a more pronounced inf lammatory response than metal surfaces (6), thus delaying rather than preventing restenosis (7,8).Polymer-coated gene-delivery stents have been demonstrated in animal studies to be effective for both reporter (9-13) and therapeutic (14, 15) vector delivery. Nevertheless, their use is problematic because of harmful properties of the polymer coatings (6, 7). Therefore, the present experiments investigated gene deli...