Michael C. Granger

Toward development of a surface-enhanced Raman scattering (SERS)-based cancer diagnostic immunoassay panel

Proteomic analyses of readily obtained human fluids (e.g., serum, urine, and saliva) indicate that the diagnosis of complex diseases will be enhanced by the simultaneous measurement of multiple biomarkers from such samples. This paper describes the development of a nanoparticle-based multiplexed platform that has the potential for simultaneous read-out of large numbers of biomolecules. For this purpose, we have chosen pancreatic adenocarcinoma (PA) as a test bed for diagnosis and prognosis. PA is a devastating form of cancer in which an estimated 86% of diagnoses resulted in death in the United States in 2010. The high mortality rate is due, in part, to the asymptomatic development of the disease and the dearth of sensitive diagnostics available for early detection. One promising route lies in the development of a serum biomarker panel that can generate a signature unique to early stage PA. We describe the design and development of a proof-of-concept PA biomarker immunoassay array coupled with surface-enhanced Raman scattering (SERS) as a sensitive readout method.

Giant Magnetoresistance Sensors. 1. Internally Calibrated Readout of Scanned Magnetic Arrays

This paper describes efforts aimed at setting the stage for the application of giant magnetoresistance sensor (GMRs) networks as readers for quantification of biolytes selectively captured and then labeled with superparamagnetic particles on a scanned chip-scale array. The novelty and long-range goal of this research draws from the potential development of a card-swipe instrument through which an array of micrometer-sized, magnetically tagged addresses (i.e., a sample stick) can be interrogated in a manner analogous to a credit card reader. This work describes the construction and testing of a first-generation instrument that uses a GMR sensor network to read the response of a simulated sample stick. The glass sample stick is composed of 20-nm-thick films of permalloy that have square or rectangular lateral footprints of up to a few hundred micrometers. Experiments were carried out to gain a fundamental understanding of the dependence of the GMR response on the separation between, and planarity of, the scanned sample stick and sensor. Results showed that the complex interplay between these experimentally controllable variables strongly affect the shape and magnitude of the observed signal and, ultimately, the limit of detection. This study also assessed the merits of using on-sample standards as internal references as a facile means to account for small variations in the gap between the sample stick and sensor. These findings were then analyzed to determine various analytical figures of merit (e.g., limit of detection in terms of the amount of magnetizable material on each address) for this readout strategy. An in-depth description of the first-generation test equipment is presented, along with a brief discussion of the potential widespread applicability of the concept.

Giant magenetoresistive sensors. 2. Detection of biorecognition events at self-referencing and magnetically tagged arrays

Microfabricated devices formed from alternating layers of magnetic and nonmagnetic materials at combined thicknesses of a few hundred nanometers exhibit a phenomenon known as the giant magnetoresistance effect. Devices based on this effect are known as giant magnetoresistive (GMR) sensors. The resistance of a GMR is dependent on the strength of an external magnetic field, which has resulted in the widespread usage of such platforms in high-speed, high-data density storage drives. The same attributes (i.e., sensitivity, small size, and speed) are also important embodiments of many types of bioanalytical sensors, pointing to an intriguing opportunity via an integration of GMR technology, magnetic labeling strategies, and biorecognition elements (e.g., antibodies). This paper describes the utilization of GMRs for the detection of streptavidin-coated magnetic particles that are selectively captured by biotinylated gold addresses on a 2 x 0.3 cm sample stick. A GMR sensor network reads the addresses on a sample stick in a manner that begins to emulate that of a card-swipe system. This study also takes advantage of on-sample magnetic addresses that function as references for internal calibration of the GMR response and as a facile means to account for small variations in the gap between the sample stick and sensor. The magnetic particle surface coverage at the limit of detection was determined to be approximately 2%, which corresponds to approximately 800 binding events over the 200 x 200 microm capture address. These findings, along with the potential use of streptavidin-coated magnetic particles as a universal label for antigen detection in, for example, heterogeneous assays, are discussed.

Competitive surface-enhanced Raman scattering assay for the 1,25-dihydroxy metabolite of vitamin D3

This paper describes the development and preliminary testing of a competitive surface-enhanced Raman scattering (SERS) immunoassay for calcitriol, the 1,25-dihydroxy metabolite (1,25-(OH)(2)-D(3)) of vitamin D(3). Deficiencies in 1,25-(OH)(2)-D have been linked to renal disease, while elevations are linked to hypercalcemia. Thus, there has been a sharp increase in the clinical demand for measurements of this metabolite. The work herein extends the many attributes of SERS-based sandwich immunoassays that have been exploited extensively in the detection of large biolytes (e.g., DNA, proteins, viruses, and microorganisms) into a competitive immunoassay for the low level determination of a small biolyte, 1,25-(OH)(2)-D(3) (M(w) = 416 g mol(-1)). The assay uses surface modified gold nanoparticles as SERS labels, and has a dynamic range of 10-200 pg mL(-1) and a limit of detection of 8.4 ± 1.8 pg mL(-1). These analytical performance metrics match those of tests for 1,25-(OH)(2)-D(3) that rely on radio- or enzyme-labels, while using a much smaller sample volume and eliminating the disposal of radioactive wastes. Moreover, the SERS-based data from pooled-patient sera show strong agreement with that from radioimmunoassays. The merits and potential utility of this new assay are briefly discussed.

Silica Encapsulation of Ferrimagnetic Zinc Ferrite Nanocubes Enabled by Layer-by-Layer Polyelectrolyte Deposition

Stable suspensions of magnetic nanoparticles (MNPs) with large magnetic moment, m, per particle have tremendous utility in a wide range of biological applications. However, because of the strong magnetic coupling interactions often present in these systems, it is challenging to stabilize individual, high-moment, ferro- and ferrimagnetic nanoparticles. A novel approach to encapsulate large, that is, >100 nm, ferrimagnetic zinc ferrite nanocubes (ZFNCs) with silica after an intermediary layer-by-layer polyelectrolyte deposition step is described in this paper. The seed ZFNCs are uniform in shape and size and have high saturation mass magnetic moment (σ(s) ∼100 emu/g, m ∼ 4 × 10(-13) emu/particle at 150 Oe). For the MNP system described within, successful silica encapsulation and creation of discrete ZFNCs were realized only after depositing polyelectrolyte multilayers composed of alternating polyallylamine and polystyrenesulfonate. Without the intermediary polyelectrolyte layers, magnetic dipole-dipole interactions led to the formation of linearly chained ZFNCs embedded in a silica matrix. Characterization of particle samples was performed by electron microscopy, energy-dispersive X-ray spectroscopy, infrared spectroscopy, powder X-ray diffraction, dynamic light scattering (hydrodynamic size and ζ-potential), and vibrating sample magnetometry. The results of these characterizations, which were performed after each of the synthetic steps, and synthetic details are presented.

Colloidally Assembled Zinc Ferrite Magnetic Beads: Superparamagnetic Labels with High Magnetic Moments for MR Sensors

Magnetic particles are widely used as labels in magnetoresistive sensors. To use magnetic particles as labels, several important characteristics should be considered, such as superparamagnetism, a high magnetic moment per particle (m), facile surface functionalization and biomolecule immobilization, colloidal stability, and analyte specificity. In this paper, we describe the preparation of magnetic labels with a high m, using colloidal assemblies of superparamagnetic zinc ferrite nanoparticles (ZFNPs, ∼9 nm). Also, several properties of these particles are compared with those of commercially available magnetic beads, Dynabeads and TurboBeads. The colloidally assembled zinc ferrite magnetic beads (ZFMBs, ∼160 nm) were synthesized by assembling ZFNPs via an emulsion-based assembly approach. While retaining superparamagnetism at room temperature, the m of ZFMBs is ∼4000× higher than that of the constituent ZFNPs. Surface functionalization with a layer of polyacrylic acid stabilized the ZFMBs in aqueous solution and enabled conjugation with streptavidin via carbodiimide linking chemistry. The streptavidinated ZFMBs can be suspended in aqueous buffer for ≥24 h, whereas 1.05 μm Dynabeads and 30 nm TurboBeads undergo ballistic deposition and instantaneous aggregation in solution, respectively. Finally, the streptavidinated ZFMBs were employed as labels in an immunoassay for the detection of osteopontin, a potential pancreatic cancer marker, proving superior to the commercial particles in terms of limit of detection and dynamic range. We expect that the work presented in this article can be extended to other biological applications, especially where superparamagnetic particles with a high m and colloidal stability are needed.

Frequency-Domain Approach To Determine Magnetic Address-Sensor Separation Distance Using the Harmonic Ratio Method.

In this work, we describe an approach to determine the distance separating a magnetic address from a scanning magnetoresistive sensor, a critical adjustable parameter for certain bioassay analyses where magnetic nanoparticles are used as labels. Our approach is leveraged from the harmonic ratio method (HRM), a method used in the hard drive industry to control the distance separating a magnetoresistive read head from its data platter with nanometer resolution. At the heart of the HRM is an amplitude comparison of a signals fundamental frequency to that of its harmonics. When the signal is derived from the magnetic field pattern of a periodic array of magnetic addresses, the harmonic ratio contains the information necessary to determine the separation between the address array and the read head. The elegance of the HRM is that there is no need of additional components to the detection platform to determine a separation distance; the streaming bit signal contains all the information needed. In this work, we demonstrate that the tenets governing HRM used in the hard drive industry can be applied to the bioanalytical arena where submicrometer to 100 μm separations are required.

Design, characterization, and integration of nanometric objects with chip-scale platforms for disease diagnosis

Nanomaterials (e.g., metal nanoparticles) are playing increasingly important roles in disease detection. This emergence arises from the need to detect markers and pathogens at ever-lower levels in human and veterinary diagnostics, homeland security, and food and water. This paper reviews our recent work using surface enhanced Raman scattering for detection of proteins, viruses, and microorganisms in heterogeneous immunoassays. It describes the assay platform, which consists of an antibody-modified capture substrate and gold nanoparticle-based label. The latter draws on the ability to reproducibly construct gold nanoparticles modified with a monolayer of an intrinsically strong Raman scatterer that is coated with a layer of antibodies. This construct, referred to as an extrinsic Raman label, exploits both the signal enhancement of scatterers when coated on nanometer-sized gold particles and the antigenic binding specificity of the immobilized antibody layer. Issues related to nonspecific adsorption, particle stability, and measurement reproducibility are also discussed.

Calibrant-free analyte quantitation via a variable velocity flow cell

In this paper, we describe a novel method for analyte quantitation that does not rely on calibrants, internal standards, or calibration curves but, rather, leverages the relationship between disparate and predictable surface-directed analyte flux to an array of sensing addresses and a measured resultant signal. To reduce this concept to practice, we fabricated two flow cells such that the mean linear fluid velocity, U, was varied systematically over an array of electrodes positioned along the flow axis. This resulted in a predictable variation of the address-directed flux of a redox analyte, ferrocenedimethanol (FDM). The resultant limiting currents measured at a series of these electrodes, and accurately described by a convective-diffusive transport model, provided a means to calculate an unknown concentration without the use of calibrants, internal standards, or a calibration curve. Furthermore, the experiment and concentration calculation only takes minutes to perform. Deviation in calculated FDM concentrations from true values was minimized to less than 0.5% when empirically derived values of U were employed.

Nanomaterial strategies for immunodetection

Metallic nanoparticles are playing increasingly important roles in biodiagnostic platforms. This emergence reflects the need to detect disease indicating entities at increasingly lower levels in human and veterinary diagnostics, homeland security, and food and water safety. To establish this perspective, this paper overviews our recent work using surface enhanced Raman scattering for detection of proteins, viruses, and microorganisms in heterogeneous immunoassays. It describes the assay platform, which is comprised of an antibody-modified capture substrate and gold nanoparticle-based label. The latter draws on the ability to reproducibly construct gold nanoparticles modified with a monolayer of an intrinsically strong Raman scatterer that is then coated with a layer of antibodies. This construct, referred to as an extrinsic Raman label, takes advantage of the signal enhancement of scatterers when coated on nanometer-sized gold particles and the antigenic binding specificity of the immobilized antibody layer. Challenges related to nonspecific adsorption, particle stability, and measurement reproducibility are also briefly examined.