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POINT-OF-CARE TECHNOLOGIES

Figure 1. (click to enlarge) Characteristic M = M(H) for a superparamagnetic material.

The advantages of using magnetic beads as labels in bioassays have been well established. Magnetic beads are not affected by reagent chemistry or photobleaching and are therefore stable over time. In addition, the magnetic background in a biomolecular sample is usually insignificant, and magnetic beads can be manipulated remotely by magnetism.

A number of magnetic field detection devices have been developed for various biosensing applications (e.g., giant magnetoresistive (GMR) sensors and spin valves, piezoresistive cantilevers, inductive sensors, superconducting quantum interference devices (SQUID), anisotropic magnetoresistive rings, and miniature Hall crosses). However, for numerous reasons, none of these detection methods are widely used in IVD systems. Either they are incorporated in instruments that feature moving parts, or they require expensive consumables, embedded electronics, liquid-nitrogen refrigeration, connections, or microfluidics.

This article discusses an alternative approach to magnetic field detection. MIAtek by Magnisense (Paris) is a technology that utilizes a unique quality of magnetic beads to deliver a simple, high-performance point-of-care (POC) solution. The MIAtek technology uses the intrinsic ability of a magnetic field to pass through plastic, water, nitrocellulose, and other materials, thereby providing true volumetric measurements. The technology also enables the development of rapid tests in multiple formats that are explored in further detail below.

Even though a rapid POC diagnostic would be useful in many applications, current technologies have not provided for the development of a simple test with adequate performance for a target of interest. As an alternative to POC diagnostic technologies, MIAtek enables a higher level of test performance in a simple test format.

Specific Measurements of Nonlinear Magnetic Signals

MIAtek is a patented technology that exploits the unique nonlinear magnetic signals of superparamagnetic materials embedded in magnetic beads (see Figure 1). Unlike conventional methods that measure the susceptibility (χ) of superparamagnetic materials, MIAtek measures only the nonlinear impact on a magnetic field that is applied by a reader (β). This process eliminates the contribution of linear diamagnetic or paramagnetic materials such as the sample matrix, consumable plastics, and nitrocellulose.

The intrinsic magnetic noise generated by the MIAtek reader is very low, with typical susceptibility values of –10^–5 (dia) or +10^–3 (para). Nevertheless, when investigating very small quantities of superparamagnetic materials (e.g., nanograms per test), the background signal generated by ancillary materials cannot be ignored.

While the amount of magnetic material of interest is very small compared with the water, plastic, and nitrocellulose in the samples and consumables, by accessing the material directly (β), background signals are eliminated. Furthermore, the effects of external magnetic fields (e.g., the earth field, motors, or magnets in a laboratory) are completely removed.

Multifrequency Field Modulation

Figure 2. (click to enlarge) The principle of magnetic bead detection by nonlinear response at combinatorial frequencies.

In magnetic immunoassays, magnetic beads are exposed to an alternating magnetic field at two frequencies: f₁ and f₂ (see Figure 2).^1,2 The amplitude of the field component with the lower frequency f₂ can be made high enough to magnetize the beads that are close to saturation. The component periodically turns on and off the ability to magnetize the beads further. When magnetic beads can be further magnetized, the higher-frequency component-f₁, will contribute to the resulting induction signal.

As a result, both frequencies will modulate the response voltage of the reading coil. MIAtek measures the response at combinatorial frequencies (e.g., f = f₁ ± 2·f₂). Tuning f₁, f₂, and their amplitudes will optimize the signal-to-noise ratio for each magnetic material. Using only one excitation frequency and recording the response at the harmonics of this frequency is also possible.

Figure 3. (click to enlarge) Fourier spectra of magnetic beads submitted to an alternating field obtained with MIAtek technology. * SPM - superparamagnetic

However, for biosensing, the signal-to-noise ratio of f is much higher for two ac frequencies, which allows MIAtek to measure a relative change of magnetic susceptibility at a level of up to 10^–8 and use this principle for magnetic-bead counting. The response at the combinatorial frequency is proportional to the amount of nonlinear magnetic material or the number of magnetic beads, providing the beads have a small size variance (see Figure 3).

Methods that submit magnetic beads to an alternating monofrequency field measure a small variation of a strong signal, which is very difficult to discern. In contrast, measuring the appearance of an entirely new signal in the spectrum enriched by intermodulation is easier.

Lowered Engineering Constraints

Magnisense has developed a reader instrument with reduced engineering constraints for the MIAtek technology. With a reading head based on simple copper coils, this instrument does not require any moving parts. In addition, because it provides true volumetric reading capability and geometric versatility, the instrument can accommodate a variety of assay formats such as lateral-flow strip tests, large-volume cartridges that combine immunoconcentration and volumetric detection, and flow systems for continuous multiagent monitoring.

Microarrays and planar biochips can also be noninvasively read or scanned by MIAtek using planar coils, which enables many application possibilities. So far, two families of consumables have been developed for this reader: MIAstrip, which is based on a lateral-flow membrane; and MIAflo, which is based on a three-dimensional filter as the reactive zone.

Reader Detection

Figure 4. The MIAtek tabletop reader by Magnisense.

The MIAtek technology is implemented with a portable reader featuring a personal digital assistant interface that can be connected to a personal computer, a laptop computer, or a laboratory information management system. Other smaller, handheld prototypes that use 1.5-V batteries have also been evaluated and are comparable in sensitivity to the tabletop version (see Figure 4).

The reader can detect 1000 MyOne beads or 100 Dynabead M-280 beads by Invitrogen (Carlsbad, CA). Such detection equates to 3 ng of Fe₃O₄ nanoparticles in a volume of 0.1 cm³ with a highly linear dynamic measurement range of almost five orders of magnitude.

Implementation in MIAflo

Figure 5. The MIAflo test cartridge by Magnisense.

The MIAflo device combines immunoconcentration and detection in the same single-dose cartridge (see Figure 5). Porous filters are used as a media for immunoconcentration, filtration of the sample, and as the solid phase for the assay. The reactive zone is a three-dimensional filter that is 2.6–5.5 mm in diameter and 3–7 mm long, and is sealed inside a tip or directly in a syringe. In this sandwich immunoassay format, the filter is coated with antibodies specific for the target antigen.

The sample, magnetic reagent, and washing solutions are passed through the filter either manually with a syringe or pipette, or with any automated fluid-handling device. Preliminary results using MIAflo were obtained for the following targets: Francisella tularensis, Salmonella typhimurium, Listeria monocytogenes, Legionella pneumophila, and Yersinia pestis. For Legionella in a water sample, a detection limit as low as 103 cfu was achieved when 1 ml of sample was processed through the device, thus eliminating a time-consuming culture phase and providing results within hours.

Figure 6. (click to enlarge) Magnetic immunoassay results for different concentrations of inactivated Legionella pneumophila using the MIAflo test.

For Y. pestis and for S. typhimurium, 0.1 ng/ml and 103 cell/ml, respectively, were detected (see Figure 6). These values are better by one and two orders of magnitude, respectively, when compared with results obtained in a traditional enzyme-linked immunosorbent assay (ELISA), with the same immunoreagents and standard horseradish peroxidase–conjugated detection reagent. In addition, the magnetic assay was completed in 20–60 minutes, while an ELISA required more than four hours. The magnetic assay time can be further reduced by immobilizing the antibodies on the filters prior to running the assay.

The detection limits were defined by the nonspecific binding of magnetic beads, while the reader’s electronic noise was about two orders of magnitude lower. This limit can be improved by advanced interface chemistry or magnetic enrichment.

The reagents are presented to the MIAflo instrument in standard ELISA plates, and eight syringes operate simultaneously to deliver the reagents. A capture antibody (Ab) is immobilized on ethanol-activated filters. A 1% BSA antigen (Ag), a second tracer biotinylated antibody (Ab-biotin), and magnetic beads coated by streptavidin are then successively passed through the filters. Several washes with optimized buffers are used after each step. For each antigen concentration, the relative quantities of magnetic beads are measured by the readout device, which had electronic noise at the level of 1–2 relative units and no baseline drift after five minutes of operation. The filters with high antigen concentrations (420 ng/ml or 4 × 10⁵ cell/ml) can be inspected by optical readers or visually, since magnetic beads look like rust.

Implementation in Lateral-Flow Tests

MIAtek can upgrade conventional lateral-flow membrane tests into high-performance quantitative tests by replacing conventional conjugates (e.g., gold, colored latex) with magnetic conjugates.

For example, a sandwich assay was developed for the quantitation of antibodies to tetanus toxin in whole blood. Anatoxin was deposited at the strip’s test line. Estapor 300-nm magnetic beads by Merck-Chimie SAS (Fontenay sous Bois, France) that were coated with anatoxin were sprayed on the conjugate pad. The antitetanus antibody in the sample (World Health Organization [WHO; Geneva] sera were used) reacted with the anatoxin-coated magnetic beads on the conjugate pad. These complexes then reacted with the anatoxin at the test line where the magnetic beads accumulated.

After 15 minutes, the reader evaluated the quantity of magnetic beads accumulated at the test line and converted these data to a quantity of antibody relative to reference standards. The detection limit of sensitivity was below 0.03 IU/ml, much lower than the 0.2 IU/ml required by WHO, and the large dynamic range allowed a titration well beyond 1.0 IU/ml. Rapid access to such quantitative results can help clinicians make proper decisions for treating patients.

Another assay format was investigated for detecting the avian flu virus in poultry tracheal and rectal samples. The conjugate pad and test line were both coated with antibodies specific for the type A flu virus. The conjugate end of the strip was inserted in a tube containing 300 µl of a mixture of sample and extraction buffer.

This solution migrated along the strip for 10 minutes and was analyzed by the reader. A full range of dilutions of a vaccine strain were tested. Despite limited optimization (no optimization of the membrane itself), the detection limit was approximately tenfold lower than conventional gold-particle-based tests, and the dynamic range for quantitation was at least two orders of magnitude greater.

In addition to its dynamic capabilities, MIAtek can accelerate assay development by establishing precise levels of materials across the reaction strip for the duration of the reaction. Sample and reagent migration along the strip can be monitored with a simple elevator device fitted to the reader and the ad hoc software. This capability can increase the signal-to-noise ratio, rather than focusing only on the increase of the raw signal, and can accelerate the speed of development of new tests.

New IVD Capabilities

The full potential of the MIAtek technology is likely to be far greater than single monoparametric IVD tests, since the use of magnetic beads in multiparametric assays has been demonstrated. By using a set of magnetic beads that differ in their magnetic properties, a single sample containing a plurality of targets can be analyzed, and each target can be independently quantified. Several families of magnetic materials with unique signatures have already been identified. This technology can be applied in several formats, including the MIAflo cartridge or other lateral-flow devices.

A device that can read planar biochips or microfluidic cartridges by using a number of small coils with a 0.5–1-mm radius is also being developed. Such coils can count magnetic nanoparticles inside a semisphere of the same radius, which allows the separation of electronics from biochips or microfluidic cartridges that can be analyzed externally through 0.1-mm-thick glass or plastic. Such magnetic biochips can be very affordable consumables for clinical diagnostics.

Eventually, the MIAtek technology will be considered a useful tool for in vivo investigations.³ When compared with the detection of 59Fe isotope for monitoring ferrite-based particles in rat blood, preliminary experiments with MIAtek demonstrated comparable sensitivity, which could be further improved by optimizing the device geometry. This approach is more convenient, safer, less expensive, and provides real-time measurements in vivo.

Conclusion

The MIAtek technology promises to make magnetic immunoassays and other magnetic bioassays the reference standard for the IVD industry. Magnetic-bead assays utilizing MIAtek have the advantages of radio-labeling, the safety of ELISA, and new capabilities to create the next paradigm in POC test development.

MIAtek could unlock a broad range of diagnostic applications that have been untapped by current detection methods. The specific needs of such markets as bacteriology and food safety testing could be better met by MIAtek. With larger-volume samples and the need to accommodate thick or high-viscosity samples, MIAtek can determine a meaningful limit of detection where it has not previously been possible.

For bacterial detection, MIAtek is sufficiently sensitive such that in many cases, it is not necessary to culture bacteria. Thus it provides a meaningful POC result in minutes rather than days. Separation utilizing magnetic affinity and collection can be integrated into tests with simple detection formats.

The increased diagnostic performance obtained by integrating MIAtek into the common lateral-flow format increases the utility of such simple tests. For example, applications such as avian flu testing in birds are being developed. Screening tests that had been relegated to a noncritical role can become clinically important methods for enhancing healthcare delivery due to increased sensitivity, decreased demands on the users, and secure noninterpretative results. As an example, a test for determining tetanus immunity status is close to market launch in the European Union.

MIAtek could also be applied to quantitative, rapid field tests that will enable remote diagnosis by a qualified medical practitioner, even though the test operator may not be medically trained. In the developing world, such remote diagnosis translates into the ability to screen vast low-population areas from a small number of centrally located clinics. In developed countries, such as the United States with its requirements to receive a waiver under the Clinical Laboratory Improvement Amendments (CLIA), MIAtek enables the development of clinically critical tests that can be administered by technicians and nurses without involving a physician.

In addition, MIAtek will elevate the rapid test beyond immunoassays by opening up new opportunities for integrating complex protocols into simple two- or three-step procedures. For example, integrated antibody separation, magnetic separation, magnetic concentration, DNA hybridization, and detection can be accomplished in the same phase. This ability offers many interesting opportunities to move the lab closer to the patient.

Finally, low concentrations of iron oxide have been shown to be nontoxic, which opens up the potential for in vivo imaging. Experiments have demonstrated that a closed circulatory loop passing through a reader permits real-time diagnostic analysis of a patient. Quantified multiparametric magnetic staining is also a possibility in which a spatial concentration of particular antibodies or DNA probes can be not only identified but also quantified on a slide. This technology offers a simple platform for advanced next-generation diagnostics.

Luc Lenglet is president of Magnisense (Paris). He can be reached at luc.lenglet@ magnisense.com.	Petr Nikitin, PhD, is chief scientific officer at Magnisense. He can be reached at petr.nikitin@ magnisense.com.	Clayton Péquignot is director of business development at Magnisense. He can be reached at clay.pequignot@ magnisense.com.