Takako Mizoguchi

Fast Detection of Biological Targets With Magnetic Marker and SQUID

We have been developing a SQUID system for the detection of biological targets. In this system, magnetic markers are bound to the targets, and the magnetic signal from the bound markers is detected with the SQUID. In order to realize fast detection of the targets, we developed a liquid-phase detection method. First, we used large polymer beads as material to capture the targets. Since the polymer beads are uniformly dispersed in liquid, biological targets on the surface of the polymer bead can be easily coupled to the markers, which results in the fast reaction time. Next, we detected the bound markers without using the washing process to separate the bound and unbound markers, which was realized by using the difference in the Brownian relaxation time between them. Using this procedure, we demonstrated the detection of the target called IgE, as well as biotin-coated polymer beads. We obtained a good relationship between the amount of IgE and the magnetic signal. The result was the same as that obtained using the conventional procedure. The reaction time for the coupling between the magnetic marker and the target was 4 min, which was much shorter than the conventional method. These results show the usefulness of the present method.

Liquid Phase Immunoassay Using AC Susceptibility Measurement of Magnetic Markers

AC susceptibility measurement of magnetic markers in solution was applied to biological immunoassays. In this method, the markers that bound to biological target can be detected in the presence of unbound markers. The bound markers were distinguished from the unbound ones by using their different frequency dependence of susceptibility. We detected biotin-conjugated polymer beads with avidin-coated Fe3O4 markers. Changes of the susceptibility caused by the binding reaction between them were shown. Good relationship was obtained between the decrease of susceptibility and the number of polymer beads. Sensitivity of the system was estimated as 3×10-16 mol/ml in terms of molecular-number concentration.

Liquid phase immunoassays utilizing magnetic markers and SQUID magnetometer.

Abstract Background: Immunoassays are one main detection system used in the field of clinical chemistry. Recent developments of a new detection method utilizing a magnetic marker and magnetic sensor have enabled rapid and sensitive immunoassay without the need for bound/free (BF) separation. Methods: Newly-synthesized conjugated avidin was used as the magnetic marker for quantitative analysis of human interleukin-8 (hIL-8) and immunoglobulin E (hIgE) in several media. A superconducting quantum interference device sensor detected the magnetic fields from markers fixed to antigens by the sandwich method. Magnetic signals from unbound markers were nearly zero due to Brownian rotation. Results: Our magnetic immunoassay could detect four attomoles of model proteins (hIL-8, hIgE) in phosphate buffer without BF separation. Using our standard curve, the range of protein detected ranged from 40 femtomoles to 4 attomoles, and we observed a strong association between protein amounts and magnetic signals from the bound markers. The homogeneous immunoassay could also quantify three hundred cells from the fungus Candida albicans in phosphate buffer. Conclusions: The present study demonstrates the ability of magnetic markers for measuring biological targets without BF separation. This detection system has great potential for use as the next generations analytical system. Clin Chem Lab Med 2010;48:1263–9.

Development of a SQUID System Using Field Reversal for Rapidly Detecting Bacteria

Pathogen identification usually requires growth of the pathogen by culture, which requires considerable time and manipulation by an experienced operator, leading to delays in diagnosis and treatment. We have investigated pathogen detection using a highly sensitive HTS-SQUID and magnetic markers and have developed a rapid and simple pathogen detection method. The magnetic markers, magnetic nanoparticles coated with detecting antibodies, bind to the target substance (antigen). The magnetic signal of the bound markers is measured with the highly sensitive SQUID. A remarkable feature of the magnetic assay is the disappearance of the magnetic signal from the unbound markers due to Brownian rotation. This makes it possible to detect the magnetic signal of the bound magnetic markers without removal of the unbound markers. In practice, however, the residual field around the SQUID generates an undesired magnetic signal from the unbound markers as a result of biased Brownian rotation. We developed a field reversal method - a measurement scheme - that eliminates the magnetic signal from the unbound markers. A difference signal is obtained by subtracting the magnetic signals measured by applying a magnetization field in two directions. The validity of this method was demonstrated experimentally using polymer beads as simulated bacteria. Its feasibility was demonstrated by the detection of Candida albicans, a pathogenic fungus. A magnetic signal of 3 mPhi0 was detected from a sample containing 300 cells of Candida albicans. The detection limit was estimated from the system noise level of 0.5 mPhi0 to be about one hundred cells of Candida albicans, indicating that this method has high sensitivity. These results show that magnetic assay using a highly sensitive SQUID can provide rapid and simple pathogen testing without culture.

Liquid-phase immunoassay utilizing binding reactions between magnetic markers and targets in the presence of a magnetic field

We developed a new and improved method for the liquid-phase detection of biological targets using magnetic markers. Unlike conventional studies, we performed a binding reaction between markers and targets in the presence of a magnetic field. This field acts to prevent the Brownian rotation of markers during the reaction. In this case, markers are bound to the targets with their magnetic moments (m?s) aligned, which is in contrast to the conventional case where m?s are randomly oriented after the reaction. As a result, we could obtain much larger signals from the bound markers without increasing the blank signal from the free markers.

Highly sensitive third-harmonic detection method of magnetic nanoparticles using an AC susceptibility measurement system for liquid-phase assay

Magnetic sensing techniques have been recently developed to detect biomarkers using magnetic nanoparticles (MNPs) in liquid-phase assays. In the case of alternating current (ac) magnetic susceptibility when detecting a low number of MNPs, the diamagnetic signal of water becomes a major problem at a low signal-to-noise ratio. Therefore, we developed a high-temperature superconducting-quantum-interference-device-based ac susceptibility measurement system that can detect third-harmonic signals from MNPs. On the basis of the nonlinear characteristic of MNPs with large ac magnetic excitation (1.06 kHz), the third-harmonic signal detection (3.18 kHz) of MNPs leads to the elimination of the diamagnetic signal of water. The system consists of excitation and gradiometer pickup coils and a multifunctional circuit device. To improve the signal-to-noise ratio of the third-harmonic measurement, the system has two cancelation functions regarding the fundamental magnetic field. The MNPs are magnetized by using the fundamental magnetic field using the excitation coil. Third-harmonic signals from the MNPs were then detected during the up-and-down movement of the MNPs. We evaluated the detection sensitivity of our system using MNPs. We confirmed that the limit of detection consistency of MNPs was 10 ng by using the third-harmonic measurement method.

Immunoassay detection without washing by using AC magnetic susceptibility

Our aim in this study was to demonstrate a stable immunoassay using the decrease of AC magnetic susceptibility without washing to separate bound and unbound magnetic markers. To achieve low noise in the immunoassay system, we examined the arrangement of the MR sensors, the calibration of the distance between the MR sensor and the sample, and the magnetic noise generated from the motor. The immunoassay system was improved based on these factors, and we detected mouse IgG conjugated polymer beads with anti-mouse IgG antibody-coated magnetic markers. The noise that resulted from the vibration of the MR sensor was decreased to about 1/6 by fixing the MR sensor on the excitation coils. The difference in the magnetic signal from each sample was also decreased from 40 % to 6% by calibrating the distance. Magnetic signal with a high signal-to-noise ratio (SN = 10 or more) was obtained by low-speed rotation (8 rpm) of the sample and the narrow band (5.3 Hz) of the lock-in amplifier demodulating the magnetic signal. The decrease of the AC magnetic susceptibility showed a strong correlation with the concentration of mouse IgG. Sensitivity of the immunoassay system could be estimated as about 4 fmol/ml in terms of molecular concentration. Immunoassay detection using AC magnetic susceptibility enabled a highly sensitive immunoassay without the conventional washing process.

Improvement of immunoassay detection system by using alternating current magnetic susceptibility

A major goal with this research was to develop a low-cost and highly sensitive immunoassay detection system by using alternating current (AC) magnetic susceptibility. We fabricated an improved prototype of our previously developed immunoassay detection system and evaluated its performance. The prototype continuously moved sample containers by using a magnetically shielded brushless motor, which passes between two anisotropic magneto resistance (AMR) sensors. These sensors detected the magnetic signal in the direction where each sample container passed them. We used the differential signal obtained from each AMR sensors output to improve the signal-to-noise ratio (SNR) of the magnetic signal measurement. Biotin-conjugated polymer beads with avidin-coated magnetic particles were prepared to examine the calibration curve, which represents the relation between AC magnetic susceptibility change and polymer-bead concentration. For the calibration curve measurement, we, respectively, measured the magnetic signal caused by the magnetic particles by using each AMR sensor installed near the upper or lower part in the lateral position of the passing sample containers. As a result, the SNR of the prototype was 4.5 times better than that of our previous system. Moreover, the data obtained from each AMR sensor installed near the upper part in the lateral position of the passing sample containers exhibited an accurate calibration curve that represented good correlation between AC magnetic susceptibility change and polymer-bead concentration. The conclusion drawn from these findings is that our improved immunoassay detection system will enable a low-cost and highly sensitive immunoassay.

Battery-Powered Wireless Flux-Locked Loop Circuit for Operating High-Tc SQUID

A battery-operated wireless flux-locked-loop (FLL) circuit for operating a high-Tc superconducting quantum interference device (SQUID) was developed and tested. This wireless battery-type FLL circuit consists of an FLL unit and a PC interface. A local area network with the TCP-IP protocol for wireless mode was used for communication between the PC interface and a controlling PC. The battery unit can power the FLL circuits for four hours. The FLL circuit had two bias modes: ac and dc. When the wireless-battery-type FLL circuit was implemented in a SQUID, the system noise generated by a wireless network became intrinsic high-Tc SQUID noise. Also, a 1/f noise was reduced in the case of the ac-bias-current mode. Consequently, the wireless-battery-type FLL circuit can operate a high-Tc SQUID with high sensitivity.