Epigenetic modifications, in particular DNA methylation, are gaining increasing interest as complementary information to DNA mutations for cancer diagnostics and prognostics. We introduce a method to simultaneously profile DNA mutation and methylation events for an array of sites with single site specificity. Genomic (mutation) or bisulphite-treated (methylation) DNA is amplified using nondiscriminatory primers, and the amplicons are then hybridized to a giant magnetoresistive (GMR) biosensor array followed by melting curve measurements. The GMR biosensor platform offers scalable multiplexed detection of DNA hybridization, which is insensitive to temperature variation. The melting curve approach further enhances the assay specificity and tolerance to variations in probe length. We demonstrate the utility of this method by simultaneously profiling five mutation and four methylation sites in human melanoma cell lines. The method correctly identified all mutation and methylation events and further provided quantitative assessment of methylation density validated by bisulphite pyrosequencing.Keywords: DNA array; GMR biosensor; melanoma; melting curve; methylation; mutation;

Autoantibodies are a hallmark of autoimmune diseases such as lupus and have the potential to be used as biomarkers for diverse diseases, including immunodeficiency, infectious disease, and cancer. More precise detection of antibodies to specific targets is needed to improve diagnosis of such diseases. Here, we report the development of reusable peptide microarrays, based on giant magnetoresistive (GMR) nanosensors optimized for sensitively detecting magnetic nanoparticle labels, for the detection of antibodies with a resolution of a single post-translationally modified amino acid. We have also developed a chemical regeneration scheme to perform multiplex assays with a high level of reproducibility, resulting in greatly reduced experimental costs. In addition, we show that peptides synthesized directly on the nanosensors are approximately two times more sensitive than directly spotted peptides. Reusable peptide nanosensor microarrays enable precise detection of autoantibodies with high resolution and sensitivity and show promise for investigating antibody-mediated immune responses to autoantigens, vaccines, and pathogen-derived antigens as well as other fundamental peptide–protein interactions.Keywords: autoantibody; giant magnetoresistance; lupus; nanosensors; peptide microarray; regeneration;

•Two denaturing strategies for detection of double-stranded DNA products are compared.•Binding is monitored using magnetic nanoparticle labels on GMR sensor arrays.•Dynamic detection range for single-stranded DNA covers 40 pM to 40 nM.•Heat denaturation provides fast readout with signal limited by competing strand.•Magnetic column separation provides high signal at a rate limited by particle diffusion.Microarrays and other surface-based nucleic acid detection schemes rely on the hybridization of the target to surface-bound detection probes. We present the first comparison of two strategies to detect DNA using a giant magnetoresistive (GMR) biosensor platform starting from an initially double-stranded DNA target. The target strand of interest is biotinylated and detected by the GMR sensor by linking streptavidin magnetic nanoparticles (MNPs) to the sensor surface. The sensor platform has a dynamic detection range from 40 pM to 40 nM with highly reproducible results and is used to monitor real-time binding signals. The first strategy, using off-chip heat denaturation followed by sequential on-chip incubation of the nucleic acids and MNPs, produces a signal that stabilizes quickly but the signal magnitude is reduced due to competitive rehybridization of the target in solution. The second strategy, using magnetic capture of the double-stranded product followed by denaturing, produces a higher signal but the signal increase is limited by diffusion of the MNPs. Our results show that both strategies give highly reproducible results but that the signal obtained using magnetic capture is higher and insensitive to rehybridization.

As medical and recreational use of cannabis, or marijuana, becomes more prevalent, law enforcement needs a tool to evaluate whether drivers are operating vehicles under the influence of cannabis, specifically the psychoactive substance, tetrahydrocannabinol (THC). However, the cutoff concentration of THC that causes impairment is still controversial, and current on-site screening tools are not sensitive enough to detect trace amounts of THC in oral fluids. Here we present a novel sensing platform that employs giant magnetoresistive (GMR) biosensors integrated with a portable reader system and smartphone to detect THC in saliva using competitive assays. With a simple saliva collection scheme, we have optimized the assay to measure THC in the range from 0 to 50 ng/mL, covering most cutoff values proposed in previous studies. This work facilitates on-site screening for THC and shows potential for testing of other small molecule drugs and analytes in point-of-care (POC) settings.

•Quantitative laboratory diagnostics requires trained technicians, multiple steps, and bulky instruments.•We have invented a portable quantitative immunoassay platform based on GMR technology with smartphone integration.•The platform can measure multiple protein biomarker concentrations within 15 minutes with only one-step user involvement.•Multiplex detection of human IgG and IgM antibodies with sensitivities of 0.07 and 0.33 nM, respectively, was demonstrated.Quantitative immunoassay tests in clinical laboratories require trained technicians, take hours to complete with multiple steps, and the instruments used are generally immobile–patient samples have to be sent in to the labs for analysis. This prevents quantitative immunoassay tests to be performed outside laboratory settings. A portable, quantitative immunoassay device will be valuable in rural and resource-limited areas, where access to healthcare is scarce or far away. We have invented Eigen Diagnosis Platform (EDP), a portable quantitative immunoassay platform based on Giant Magnetoresistance (GMR) biosensor technology. The platform does not require a trained technician to operate, and only requires one-step user involvement. It displays quantitative results in less than 15 min after sample insertion, and each test costs less than US$4. The GMR biosensor employed in EDP is capable of detecting multiple biomarkers in one test, enabling a wide array of immune diagnostics to be performed simultaneously. In this paper, we describe the design of EDP, and demonstrate its capability. Multiplexed assay of human immunoglobulin G and M (IgG and IgM) antibodies with EDP achieves sensitivities down to 0.07 and 0.33 nanomolar, respectively. The platform will allow lab testing to be performed in remote areas, and open up applications of immunoassay testing in other non-clinical settings, such as home, school, and office.

Circulating tumor cells (CTCs) are established cancer biomarkers for the “liquid biopsy” of tumors. Molecular analysis of single CTCs, which recapitulate primary and metastatic tumor biology, remains challenging because current platforms have limited throughput, are expensive, and are not easily translatable to the clinic. Here, we report a massively parallel, multigene-profiling nanoplatform to compartmentalize and analyze hundreds of single CTCs. After high-efficiency magnetic collection of CTC from blood, a single-cell nanowell array performs CTC mutation profiling using modular gene panels. Using this approach, we demonstrated multigene expression profiling of individual CTCs from non–small-cell lung cancer (NSCLC) patients with remarkable sensitivity. Thus, we report a high-throughput, multiplexed strategy for single-cell mutation profiling of individual lung cancer CTCs toward minimally invasive cancer therapy prediction and disease monitoring.

We demonstrate microfluidic partitioning of a giant magnetoresistive sensor array into individually addressable compartments that enhances its effective use. Using different samples and reagents in each compartment enables measuring of cross-reactive species and wide dynamic ranges on a single chip. This compartmentalization technique motivates the employment of high density sensor arrays for highly parallelized measurements in lab-on-a-chip devices.

Fabrication of high-density plasmonic dimers on a large (wafer) scale is crucial for applications in surface-enhanced spectroscopy, bio- and molecular sensing, and optoelectronics. Here, we present an experimental approach based on nanoimprint lithography and shadow evaporation that allows for the fabrication of high-density, large-scale homo- (Au–Au and Ag–Ag) and hetero- (Au–Ag) dimer substrates with precise and consistent sub-10-nm gaps. We performed scanning electron, scanning transmission electron, and atomic force microscopy studies along with a complete electron energy-loss spectroscopy (EELS) characterization. We observed distinct plasmonic modes on these dimers, which are well interpreted by finite-difference time-domain (FDTD) and plasmon hybridization calculations.Keywords: electron energy-loss spectroscopy (EELS); finite-difference time-domain (FDTD); nanoimprint lithography; plasmon hybridization; plasmonic dimers; shadow evaporation;

A double exposure technique has been used to fabricate nanoimprint stamps for making monodisperse nanorods with controllable lengths. The nanorod length is defined by a normal photolithography projection process whereas the nanorod width is defined by an edge-lithography process using a soft polydimethylsiloxane (PDMS) contact mask. Taking advantage of edge-lithography, the nanorod width can be less than the diffraction limit of the exposure light. Using these nanorod stamps, synthetic magnetic multilayer (SMM) nanorods have been fabricated using nanoimprint lithography, resulting in a length variation of ∼3%. Nanorod magnetic properties have been characterized in both longitudinal and in-plane transverse directions of the nanorods. A theoretical model has been established to explain the magnetic responses and has revealed that both shape anisotropy and interlayer interactions are important in determining the properties of SMM nanorods.

Detection and characterization of circulating tumor cells (CTCs) may reveal insights into the diagnosis and treatment of malignant disease. Technologies for isolating CTCs developed thus far suffer from one or more limitations, such as low throughput, inability to release captured cells, and reliance on expensive instrumentation for enrichment or subsequent characterization. We report a continuing development of a magnetic separation device, the magnetic sifter, which is a miniature microfluidic chip with a dense array of magnetic pores. It offers high efficiency capture of tumor cells, labeled with magnetic nanoparticles, from whole blood with high throughput and efficient release of captured cells. For subsequent characterization of CTCs, an assay, using a protein chip with giant magnetoresistive nanosensors, has been implemented for mutational analysis of CTCs enriched with the magnetic sifter. The use of these magnetic technologies, which are separate devices, may lead the way to routine preparation and characterization of “liquid biopsies” from cancer patients.

We present a resistive network model, protein assay data, and outlook of the giant magnetoresistive (GMR) spin-valve magneto-nanosensor platform ideal for multiplexed detection of protein biomarkers in solutions. The magneto-nanosensors are designed to have optimal performance considering several factors such as sensor dimension, shape anisotropy, and magnetic nanoparticle tags. The resistive network model indicates that thinner spin-valve sensors with narrower width lead to higher signals from magnetic nanoparticle tags. Standard curves and real-time measurements showed a sensitivity of ~10 pM for phosphorylated-structural maintenance of chromosome 1 (phosphor-SMC1), ~53 fM for granulocyte colony stimulation factor (GCSF), and ~460 fM for interleukin-6 (IL6), which are among the representative biomarkers for radiation exposure and cancer.

Early detection and targeted therapy are two major challenges in the battle against cancer. Novel imaging contrast agents and targeting approaches are greatly needed to improve the sensitivity and specificity of cancer theranostic agents. Here, we implemented a novel approach using a magnetic micromesh and biocompatible fluorescent magnetic nanoparticles (FMN) to magnetically enhance cancer targeting in living subjects. This approach enables magnetic targeting of systemically administered individual FMN, containing a single 8 nm superparamagnetic iron oxide core. Using a human glioblastoma mouse model, we show that nanoparticles can be magnetically retained in both the tumor neovasculature and surrounding tumor tissues. Magnetic accumulation of nanoparticles within the neovasculature was observable by fluorescence intravital microscopy in real time. Finally, we demonstrate that such magnetically enhanced cancer targeting augments the biological functions of molecules linked to the nanoparticle surface.Keywords: cancer targeting; fluorescent magnetic nanoparticle; fluorescent nanoparticle; magnetic nanoparticle; magnetic targeting; molecular imaging; nanoparticle theranostic agent

We report an autoassembly protein array capable of rapidly screening for aberrant antibody−antigen binding events. Our technique combines magnetic nanoparticle technology with proximity-based, magnetically responsive nanosensors for rapid (under 15 min) and high-density screening of antibody cross-reactivity at sensitivities down to 50 fM in a homogeneous assay. This method will enable the identification of the precise cause of aberrant or cross-reactive binding events in an easy-to-use, rapid, and high-throughput manner.

Driven by scientific progress and economic stimulus, medical diagnostics will move to a stage in which straightforward medical diagnoses are independent of physician visits and large centralized laboratories. The future of basic diagnostic medicine will lie in the hands of private individuals. We have taken significant strides towards achieving this goal by developing an autoassembly assay for disease biomarker detection which obviates the need for washing steps and is run on a handheld sensing platform. By coupling magnetic nanotechnology with an array of magnetically responsive nanosensors, we demonstrate a rapid, multiplex immunoassay that eliminates the need for trained technicians to run molecular diagnostic tests. Furthermore, the platform is battery-powered and ultraportable, allowing the assay to be run anywhere in the world by any individual.

We demonstrate top-down synthesis of monodisperse plasmonic nanoparticles designed to contain internal Raman hot spots. Our Raman-active nanoparticles are fabricated using nanoimprint lithography and thin-film deposition and are composed of novel internal structures with sublithographic dimensions: a disk-shaped Ag core, a Petri-dish-shaped SiO2 base whose inner surface is coated with Ag film, and a sub-10 nm scale circular gap between the core and the base. Confocal Raman measurements and electromagnetic simulations show that Raman hot spots appear at the inside perimeter of individual nanoparticles and serve as the source of a 1000-fold improvement of minimum molecular detection level that enables detection of signals from a few molecules near hot spots. A multimodality version of these nanoparticles, which includes the functionality offered by magnetic multilayers, is also demonstrated. These results illustrate the potential of direct fabrication for creating exotic monodisperse nanoparticles, which combine engineered internal nanostructures and multilayer composite materials, for use in nanoparticle-based molecular imaging and detection.Keywords: magnetic; nanoparticle; plasmonic; surface-enhanced Raman scattering

Giant magnetoresistive biosensors possess great potential in biomedical applications for quantitatively detecting magnetically tagged biomolecules. Magnetic sensing does not suffer from the high background levels found in optical sensing modalities such as the enzyme linked immunosorbent assay translating into a technology with higher sensitivity. However, to reveal the full potential of these sensors and compensate for non-idealities such as temperature dependence, digital correction and calibration techniques are not only useful but imperative. Using these calibration techniques to correct for process variations and dynamic changes in the sensing environment (such as temperature and magnetic field), we are able to obtain extremely sensitive and, more importantly, reproducible results for quantifiable biomolecular reorganization. The reproducibility of the system was improved by over 3× using digital correction techniques and the sensors are made temperature independent by using a novel background correction technique.

Giant magnetoresistive biosensors are becoming more prevalent for sensitive, quantifiable biomolecular detection. However, in order for magnetic biosensing to become competitive with current optical protein microarray technology, there is a need to increase the number of sensors while maintaining the high sensitivity and fast readout time characteristic of smaller arrays (1–8 sensors). In this paper, we present a circuit architecture scalable for larger sensor arrays (64 individually addressable sensors) while maintaining a high readout rate (scanning the entire array in less than 4 s). The system utilizes both time domain multiplexing and frequency domain multiplexing in order to achieve this scan rate. For the implementation, we propose a new circuit architecture that does not use a classical Wheatstone bridge to measure the small change in resistance of the sensor. Instead, an architecture designed around a transimpedance amplifier is employed. A detailed analysis of this architecture including the noise, distortion, and potential sources of errors is presented, followed by a global optimization strategy for the entire system comprising the magnetic tags, sensors, and interface electronics. To demonstrate the sensitivity, quantifiable detection of two blindly spiked samples of unknown concentrations has been performed at concentrations below the limit of detection for the enzyme-linked immunosorbent assay. Lastly, the multiplexing capability and reproducibility of the system was demonstrated by simultaneously monitoring sensors functionalized with three unique proteins at different concentrations in real-time.

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.

We present giant magnetoresistance (GMR) spin valve sensors designed for detection of superparamagnetic nanoparticles as potential biomolecular labels in magnetic biodetection technology. We discuss the sensor design and experimentally demonstrate that as few as ∼23 monodisperse 16-nm superparamagnetic Fe3O4 nanoparticles can be detected by submicron spin valve sensors at room temperature without resorting to lock-in detection. A patterned self-assembly method of nanoparticles, based on a polymer-mediated process and fine lithography, is developed for the detection. It is found that sensor signal increases linearly with the number of nanoparticles.