Medical Device Link.

Originally Published IVD Technology April 2002

Detection Technologies

Paramagnetic-particle detection in lateral-flow assays

Paramagnetic labeling offers an alternative method for analyte detection.

Ronald T. LaBorde and Brendan O'Farrell

The idea of using magnetic nanoparticles as analyte labels is not new. Researchers in the ferrofluid field have developed a variety of technologies and intellectual properties that use magnetic particles. Ranging from a few nanometers to several hundred nanometers in diameter, these colloids have been used in a wide range of applications.

In the medical field, magnetic nanoparticles have been used in blood detoxification, biochemical separations, drug delivery, nucleic acid separation and detection, and as MRI contrast agents, and tamponades in retinal surgery.^1,2 When employed to facilitate biochemical separations, colloidal paramagnetic-particle labels utilize the ability of antibodies to link selectively the analyte of interest to the magnetic nanoparticle. Whether it is a molecule, cell fragment, or complete cell, the analyte is then "labeled" with magnetic particles.

Such particles must have the property of being superparamagnetic, meaning that they are only magnetic when placed in a very strong magnetic field. This is critical to their success as labels for separation or analyte detection. If the individual particles possessed a remnant magnetic field, each particle would act as a small dipole magnet, resulting in aggregates or chains as well as destabilization and precipitation of the colloid.

To have practical utility, each paramagnetic particle (PMP) should also have surface properties that allow antibodies or recognition units to be linked to the particles. Such conjugated particles with the appropriate recognition units are then bathed with a fluid containing the analyte. After a relatively short incubation time due to the rapid diffusion of colloidal particles and the binding energies of antibodies, the analyte is magnetically labeled, and with the application of a magnetic field, the isolation or separation is performed.

A simple extension of analyte labeling is the use of a PMP not only as a separation medium, but also as a means of analyte detection. Direct measurement of magnetic iron oxide (Fe_xO_y) labels, assaying either the metal content, electrical resistance, magnetic field, or presence of atomic force microscope cantilevers, has been attempted. Such efforts have met with limited success, each method of measurement having a specific limitation for application in a diagnostic assay.

Quantum Design (San Diego) has developed a technology that uses widely available particles and biological reagents to label and measure analytes in a way that is compatible with existing diagnostic test formats. The use of colloidal gold, latex, and carbon-particle labels in immunochromatographic tests, or lateral-flow assays, has allowed the facile substitution of magnetic-particle labels in this assay format.

Quantum Design's induction technology measures the capture or test line of such tests where magnetic particles are immobilized and concentrated after being separated from the sample matrix.³ These capture lines or zones are formed when the magnetic particles bind to ligands that have been striped onto the chromatographic membrane, typically nitrocellulose.

In a conventional assay, these lines are detected visually or by an optical instrument that measures reflectance, contrast, color change, or fluorescence. Such observations of the accumulation of particles at the test line of a membrane measure at most the top 10 µm of the substrate. Typically, the membranes that are used are a few hundred microns thick. Since the particle-labeled analyte travels not only on the surface but also within the membrane, up to 90% of the analyte is not seen by a measuring device, the eye, or an optical spectrometer. Additionally, optical measurement of analytes in biological fluids can also be difficult because of interfering substances, specular reflection, scattering, self-absorption, and quenching of signal in fluorescence-based tests.

Using a magnetic label circumvents many of these issues. First, magnetic material is not commonly found in biological matrices (hemoglobin, for instance, is not magnetic). Second, in magnetic immunochromatographic tests, the entire capture or test zone is measured, not just the surface, allowing a relatively large volume of labeled analyte to be quantified.

Technical Aspects

Figure 1. The E-core magnet with analytical membrane. (click to enlarge)

In Quantum Design's magnetic assay reader (MAR), detection is performed by exposing a membrane to a group of small rectangular coils that reside in between two ferrite E-core magnets. These two E-shaped magnets are assembled with the center legs of the two Es, which are slightly shorter than the outside legs, creating a gap when placed face to face. This center portion of the E-core magnet provides a very strong, homogenous magnetic field when the windings of conductors around the outside legs of the assembled magnet are energized (see Figure 1). When the immunochromatographic test is inserted into the reader, a motor aligns and positions the test strip in the center portion of the magnet.

The MAR technology detects the presence of localized patterns of superparamagnetic particles. The magnets produce a magnetic field of about 750 gauss, which is strong enough to completely magnetize or saturate the particles and is detected by the magnetic field sensors or coils. The signal from these sensors is then amplified and processed to return a value that indicates the quantity of magnetic particles in the analytical region.

Figure 2. Magnetic sensor arrangement. (click to enlarge)

The magnetic sensors in the MAR are arranged in a pattern that is referred to as a gradiometer, which means that adjacent sensors produce signals of opposite voltage in reaction to a uniform magnetic field of large spatial extent. When the signal from both sensors is calculated the result is ideally zero, as the signal from one sensor is equal and opposite to the signal from the other.

Figure 3. Sequential placement of analytical region and sensor during measurement. (click to enlarge)

The MAR sensor array is composed of four sensors forming two adjacent gradiometers (see Figure 2). This sensor geometry is referred to as a plus-minus-plus-minus arrangement. For any magnetic field that penetrates in the same direction, the first and third sensors produce a positive signal, while the second and fourth produce a negative signal. The signal that is measured by the instrument is the sum of the signals from all four sensors. As a thin band of magnetic particles moves across the arrangement of sensors, the signal is quantified, providing a direct measurement of the total quantity of magnetic material. The thin band of magnetic particles localized at the analytical region is similar to the colloidal gold or latex particle lines developed in a conventional immunochromatographic test.

When the analytical region is far to the left of the array, none of the sensors detect the magnetic particles, and the total resulting signal from the sensors is zero (see Figure 3a). When the analytical region is positioned over the first sensor, its magnetic environment is altered compared to the other sensors; the voltage produced no longer exactly cancels out its oppositely wound counterpart, therefore producing a net voltage or signal proportional to the mass of magnetic particles. As a result, the total signal from the sensors is positive (see Figure 3b). If the analytical region, however, is positioned over the second sensor, then the second sensor sees the field from the sample where the others do not, and as a result, the total signal from the sensors is negative (see Figure 3c). The third and fourth sensors, respectively, behave in a similar manner when presented with an adjacent analytical region.

Figure 4. Sensor response versus strip position. (click to enlarge)

The graph of the signal that is produced by the sensor array plotted against the analytical region position has a characteristic curve (see Figure 4). The amplitude of this signal is proportional to the amount of magnetic material in the analytical region. To take a measurement, the instrument moves the test strip through the sensor array while repeatedly reading and storing the signal from the sensor array.

The position and amplitude of the signal from the analytical region is calculated, recorded, and compared to an ideal curve shape using numerical curve-fitting techniques. Such curve-fitting accounts for and rejects most aspects of the signal that are not the result of presenting the analytical region in the desired geometric configuration. Signals appearing in the raw data that do not have this characteristic shape can be accounted for and rejected by the curve-fitting algorithms that are used in the MAR software.

During measurement, the field excites all of the superparamagnetic particles, even within the membrane, allowing for a sensitive and quantitative measurement of the analyte. In addition, the resolution of the detector system allows the multiplexing of several analyte regions along the analytical membrane, which provides a platform for tests measuring a panel of indicators, such as cardiac markers, drugs of abuse, environmental soil and water testing, and biological warfare agents.

Figure 5. MAR 1V benchtop development instrument by Quantum Design (San Diego).

The instrument can be the size of a paperback book, including a palm-sized processor for graphical interface and software development. A larger model may be used for benchtop development and can be run from a Windows-based PC or a laptop computer (see Figure 5). The power requirements of the mechanical drive, E-core magnet, and processor are modest enough to make practicable a portable, handheld instrument that is capable of running on batteries.

Assay Development: Practical Issues and Promise

The MAR induction technology measures the analytical region of immunochromatographic tests. Except for its method of detection, the MAR technology does not differ much from current lateral-flow assays based on visible labels such as monodisperse latex suspensions, colloidal gold sols, or colloidal carbon. The advantage of the induction technology is that any assay format currently used in lateral-flow tests can be adapted to this technology. Antigens and antibodies can be combined to form a simple rapid-test method that is applicable to a wide range of analytes in qualitative, semiquantitative, and fully quantitative assay formats, either as capture or sandwich assays (see Figure 6).

Figure 6. Schematic of a typical lateral-flow capture assay format using paramagnetic particle labels. (click to enlarge)

A number of lateral-flow assay formats on the market utilize reflectance- or fluorescence-based detection systems with some success. While these formats have advantages and disadvantages, commonly recurring issues include limited dynamic range, difficulties of reproducibility, signal quenching, light scatter or specular reflection from wet membranes, high cost of reader development, and the actual cost of the reader itself.

The MAR system has been designed and demonstrated to overcome many of these issues using a variety of magnetic particles. In typical measuring circumstances using a variety of magnetic particles, prototype designs have backgrounds of one to three millivolts, with a dynamic range of four orders of magnitude. Bare nitrocellulose membranes and most plastics have low backgrounds, with a range the same as or near the instrument background.

The reproducibility of the instrument reading a standard is better than 98%. This low error rate is obtained at the minimum detectable level of 25 pg of Fe as Fe₃O₄, with a signal-to-noise ratio of 3:1.

Figure 7. Viral-antigen PMP assay: dose response (a); reproducibility (b); stability (c). (click to enlarge)

Quantum Design has been involved in several collaborations that have shown the feasibility of using magnetic nanoparticles as labels in immunochromatographic tests. The U.S. Navy's Biological Defense Research Directorate developed five assays using such tests and magnetic labels.⁴ The analytes ranged from staph enterotoxin b to cell fragments of the Francisella tularensis bacteria. The staph enterotoxin b assay demonstrated 10 fg/ml sensitivity with a better than 3:1 signal-to-noise ratio. Detection of bacteria below 30 colony forming units/ml has also been demonstrated.

Another collaboration with Binax (Portland, ME) using a model antibody system (canine heartworm) showed the ability to produce an immunochromatographic test with 100-pg sensitivity and 4% CVs using a magnetic-label system.

Figure 8. Lateral-flow PMP myoglobin assay standard curve. Three samples were used in this experiment. (click to enlarge)

Two assay systems were also examined at BioDot Inc. (Irvine, CA) for basic feasibility. The first system was based on a common viral antigen, while the second was designed for the detection of myoglobin. Both were sandwich assay formats. Using existing materials and processing methods, the primary aim was to determine whether it is possible to generate reproducible, quantifiable results in a system with sufficient sensitivity to meet or improve upon currently available assay systems (see Figures 7 and 8).

The studies at BioDot were designed to examine a number of potential concerns surrounding this platform. These concerns centered on the sensitivity of the measurement technique and the desire to make the overall system truly quantitative.

The primary concern was with the lateral-flow format itself. Given the extreme sensitivity and reproducibility of the measurement tool, the question was whether a lateral-flow system with multiple sources of variation in materials and performance could yield a signal reproducible and accurate enough to do justice to the measurement system. An analysis of the major potential sources of variation yielded the following areas of concern: paramagnetic particle system, conjugate pad system, membrane system, reagent deposition system, and physical assay format.

Paramagnetic Particle System. The particle label used in this application must have a number of clearly defined characteristics. The particle must have negligible magnetic memory to avoid destabilization of the conjugate. The particle size distribution must be small, and the Fe₃O₄ content must be consistent and quantified to ensure signal reproducibility. The ratio of Fe₃O₄ to matrix material influences both sensitivity of the assay and the separation technique used during conjugation.

In addition, the signal that is generated in the system is proportional to the cube of the particle radius, assuming a constant Fe₃O₄ content, so even small variations in particle size can theoretically have a significant effect on signal intensity. Particle charge density must also be consistent.

In practice, several sources of consistently sized superparamagnetic particles were identified which were capable of providing particles with consistent charge densities and Fe₃O₄ content. The results were generated using 130-nm particles with an iron content in the range of 0.37–0.43 mg/ml.

Conjugate Pad System. In order to achieve consistent and adequate release of conjugate from the conjugate pad, pad pretreatment, conjugate deposition, and drying must be highly efficient and consistent. It was expected that the choice of deposition method and conjugate pad material would be of considerable importance to the success of the system, and accordingly the release characterisics of several conjugate pads were examined (see Table I).

Membrane System. The characteristics of membranes that are desirable for this system are more demanding than those used in standard lateral-flow systems. Since the reader system reads a larger volume than a visual system, consistency of the read volume is important.

Signal strength of an individual particle in the magnetic field is proportional to the square of its distance from the read head. The distances involved in this system are small, on the order of 100–200 µm, with potential variation in particle positioning relative to the read head on the order of 100 µm, which is defined by the membrane's thickness.

Theoretically, however, variation in pore size distribution (and hence the internal membrane volume read) and the position of particle accumulation within the membrane relative to the read head could lead to variation in signal strength. Thin membranes with consistent pore-size distribution are therefore ideal for this system.

Other desirable membrane characteristics include a low background magnetic signal, consistent flow characteristics (low CV in capillary rise time), and resistance to changes in flow characteristics due to membrane aging effects.

The choice of membranes for these experiments was limited to those with small pore sizes (less than 8 µm) by the necessity of maintaining an overall line thickness of 0.4 µm or less, and a restriction imposed by the geometry of the read head at the time the experiments were performed.

The membrane characteristics outlined above are desirable and will simplify the development and manufacture of consistent, quantifiable assays on this platform. Even so, theoretical problems related to particle position and consistency of read volume did not cause gross inconsistencies in the ability of the experimental systems to generate reproducible, accurate results.

Reagent Deposition System. Accurate and quantitative deposition of antibodies, antigens, and conjugates is essential to the success of a quantitative assay system. The ability to achieve a consistent quantitative deposition depends on the combination of the dispensing system chosen, the characteristics of the reagent being dispensed, the characteristics of the matrix being dispensed onto, and the environment in which the deposition is being performed.

Figure 9. XYZ 3000 platform with BioJet Quanti 3000 and AirJet Quanti 3000 dispensers by BioDot Inc. (Irvine, CA).

Reagents are best dispensed using a noncontact method to ensure no contact of metallic components with the membrane. The method should also be quantitative, to ensure reproducibility of deposition per unit length of membrane. Thin lines of consistent width are critical to the reproducibility and sensitivity of the system because of the configuration of the read heads.

For the experiments, line thicknesses of 0.4 µm were consistently generated through a choice of small-pore-size membranes, combined with dispenser selection and careful environmental control. For conjugate deposition, in order to achieve the consistency necessary, immersion techniques were not attempted. All conjugate deposition was performed using a quantitative, noncontact dispenser (see Figure 9). Data previously generated at BioDot also demonstrated the advantages of using dispensing rather than immersion in achieving consistent and efficient conjugate release from a variety of pads.

In an experiment to determine the efficiency of recovering dried conjugate from a variety of conjugate pads, a series of manufacturer-treated pads and in-house-treated pads were used. The in-house-treated pads were immersed in a solution of PMPs, and the average volume per unit length absorbed by the materials was determined gravimetrically. A dispenser was used to quantitatively dispense the same volume per unit length onto the pads. The manufacturer-treated pads were not immersed, per the manufacturer's recommendation, but had the same volume per unit length of PMP solution sprayed onto them using the dispenser. All of the pads were dried and cut into strips, and the conjugate was eluted using water. The eluted conjugate pads were recovered and dried, and a total iron analysis was performed to determine the proportion of PMPs eluted from the pads (see Table I).

A marked improvement in elution efficiency was seen when the pads were sprayed as opposed to immersed before drying. The best recovery rate for immersed samples was 66%, whereas the worst recovery rate for dispensed samples was 82%. Based on these results, a dispenser was used for all conjugate deposition applications for the PMP assay development projects.

Physical Assay Format. Due to the geometry of the read head within the MAR, the options available in terms of physical assay format are limited. Several versions of the read head are available, each with a slightly different geometry (see Figure 10).

Figure 10. Test-strip geometry for use in the MAR. Recent implementation of detector arrays now allows for up to 0.8-mm-wide striped analytical regions of capture zones with 0.5 cm between these regions. (click to enlarge)

These characteristics impose a requirement for the use of nonconventional formats for tests, primarily driven by the need for thinness in the vertical axis due to the limited space available between the heads of the E-core magnet. The thinness of the assay requires some innovation on the part of the designer, as a standard-format immunochromatographic test will not fit in the system due to the thickness of the sample, conjugate, and wicking pads. A number of innovative, proprietary strip design concepts have been developed to overcome this issue and are being tested. For the purposes of the experiments, it was necessary to use a standard assay format and remove the membrane before reading, placing it onto a thin carrier matrix before insertion into the reader.

Conclusion

The results of these experiments indicate the feasibility of generating stable assay systems with wide dynamic range, capable of producing quantitative, reproducible results. Moreover, these results were generated without making any significant alterations to currently available and practiced processing methods or materials. Future improvements in both can only lead to greater accuracy and reproducibility in the system.

As the technology develops, many existing immunochromatographic test assays that require sensitivity not easily attainable with optically read labels will be improved by using magnetic labels. In addition, the improved sensitivity and quantification which this platform potentially provides may allow for the development of immunochromatographic tests for clinically significant markers that previously could not be detected using immunochromatographic assays. As a result, many analytes that have been confined to the realm of the laboratory and clinical instrumentation will now present opportunities for the development of rapid point-of-care tests.

References

1. "Proceedings of the Third International Conference on Scientific and Clinical Applications of Magnetic Carriers," ed. U Haflei, Journal of Magnetism and Magnetic Materials 225, no. 1-2 (2001): 1–314.

2. M Zahn and K Shenton, "Magnetic Fluids Bibliography," IEEE Transactions on Magnetics 16, no. 2 (1980): 387–490.

3. MB Simmonds, Method and apparatus for making quantitative measurements of localized accumulations of target particles having magnetic particles bound thereto, U.S. Pat. 6,046,585, (2000).

4. R Bull et al., "Defense Advanced Research Projects Agency (DARPA) Broad Agency Announcement," (presented at BioMagnetics Workshop, Arlington, VA, December 11–12, 2001).

Ronald T. LaBorde is technical director of the biotechnology division at Quantum Design (San Diego). He can be reached via laborde@qdusa.com. Brendan O'Farrell is director of research and development at BioDot Inc. (Irvine, CA). He can be reached via bofarrell@biodot.com.

Photos courtesy Quantum Design
Photo courtesy Biodot Inc.

Copyright ©2002 IVD Technology