Rapid virus detection using magnetic second harmonics of superparamagnetic iron oxide nanoparticles

Rapid screening of people infected with COVID-19¹ or influenza² has become necessary. Polymerase chain reaction (PCR)³ and enzyme-linked immunosorbent assay (ELISA) are regarded as the gold standard methods for virus detection. Although their detection sensitivity is significantly high, long measurement time and high costs are their limitations. The rapid influenza diagnostic test (RIDT)⁴ has the advantage of short measurement time for influenza viruses. However, its detection accuracy is inferior to those of other methods.^2,5 Magnetic sensing using giant magnetoresistance (GMR)/tunnel magnetoresistance (TMR) sensors has the advantage of high sensitivity. However, multiple washing steps usually required, thus well-trained technicians are needed.^6–9 Magnetic particle spectroscopy (MPS), which utilizes the nonlinear magnetization response of magnetic nanoparticles (MNPs) under applied alternating current (AC) magnetic fields,^10–13 has been developed for rapid and accurate virus detection. This method does not require any washing steps or well-trained technicians. The AC magnetic signals of the MNPs that conjugate to the virus are significantly altered with increasing virus concentration. This is a novel screening method owing to its high sensitivity, short measurement time, and affordability. In particular, the third harmonics without bias magnetic fields are mainly used as an indicator for virus detection to eliminate the magnetic noise of the fundamental frequency. Applying AC and direct current (DC) bias magnetic fields to MNPs generates even-order harmonics, and the signal intensity of the second harmonic signal is higher than that of the third harmonic signal.^14–18 We previously proposed a virus detection method using second harmonics under DC magnetic fields and demonstrated that the second harmonic was a more effective indicator for virus detection than the third harmonic.¹⁹ In this study, we verified the optimal MNPs concentration for virus detection using the second harmonic and report the detection of virus-imitating polymer nanobeads by the signal-to-noise ratio (SNR) of the second and third harmonics. In addition, we demonstrated the relationship between the particle size of MNPs in liquid and the concentration of beads bound to MNPs in the virus detection system based on second harmonics.

Figure 1 shows the experimental setup of the virus detection system. The excitation coil (solid copper wire of gauge #24, 1000 windings, inner diameter is 14.1 mm, outer diameter is 28.3 mm, coil length is 40 mm) and pick-up coils (solid copper wire of gauge #32, clockwise and counterclockwise with 600 turns, inner diameter is 13.9 mm, outer diameter is 11 mm, coil length is 20 mm) are placed inside the DC bias coil (solid copper wire of gauge #17, 1200 windings, inner diameter is 55 mm, outer diameter is 160 mm, coil length is 60 mm), which generates the DC magnetic field from 0 mT to 13 mT. The DC power source (TEXIO PSW-1080H800) supplies electric current to the DC bias coil. The sine wave of the signal generator (NF Corporation WF1948) was amplified by a bipolar amplifier (NF Corporation HSA4101) and AC magnetic fields with an amplitude of 6.6 mT at 943 Hz were driven through the excitation coil. The 6.6 mT was measured using a Hall probe. We use 6.6 mT to magnetize the MNPs of samples sufficiently. The amplitudes also consider the safe current of the excitation coil for coil temperature. The excitation frequency error level is 0.1%, and the excitation current error level is 0.16%. The pick-up coils with gradiometer configuration detect the magnetization signal from MNPs. A compensation coil is used to reduce the magnetic noise in the pick-up coil. The magnetic signal is measured using a lock-in amplifier (Stanford Research Systems SR830) through a band-pass filter (NF Corporation 3628) and a resonance circuit. The frequency on the resonance circuit is switched according to the harmonic frequency and the second and third harmonic frequencies.

We employ MNPs (Synomag-D, average size of 50 nm, original MNP concentration is 5.0 mg/ml) coated with streptavidin^13,20 and polymer nanobeads (yellow fluorescent particles, average size of 60 nm) coated with a layer of biotin to imitate the virus as an alternative to the virus and bind to MNPs through avidin-biotin conjugation. We measured the signal originating from eight types of samples with different beads concentration of polymer nanobeads (10³, 10⁵, 10⁷, and 10⁹ number of beads, corresponding to 10⁻⁹, 10⁻⁷, 10⁻⁵, and 10⁻³ mg/ml concentration, respectively) bound to two types of MNP concentration (0.25 and 1.25 mg/ml, corresponding to 2 × 10¹⁰ and 10¹¹ number of MNPs). The beads concentrations of our experiment are in the detection sensitivity range of the PCR and RIDT methods, which is the practical concentration. The sample containing the MNPs and nanobeads volume was 40 µl. To calculate the SNR, we evaluated the measured voltage with the sample as the signal and the standard deviation of the measured voltage without the sample as the noise. The sample was placed at the center of the upper pick-up coil. The measurement time of the magnetization response of the MNPs is one minute for rapid virus detection. To evaluate particle aggregation, we measured the hydrodynamic size (D_H) of the MNP-bead samples by Dynamic Light Scattering (DLS) (Malvern Panalytical, Zetasizer Nano ZS). For the DLS measurement, the sample diluted four times was used.

The concentration of MNPs has a significant impact on virus detection.²¹ When the concentration of MNPs is too low, the magnetic signal of the MNPs decreases. However, when the concentration of MNPs is too high relative to the concentration of virus (nanobeads), the change in the magnetic signals becomes small with increasing virus concentration.²¹ Therefore, experiments were conducted with varying concentrations of virus to verify the virus detection accuracy. We verified how the intensity of each harmonic signal, including the second harmonic signal, changes with the application of a DC magnetic field. Figure 2 shows the DC magnetic field dependence of the magnetic signal of harmonics R/f (harmonic signal intensity normalized by its frequency) from the fundamental frequency to the third harmonic for the MNP sample. The voltage signal measured by the detection coil increases in proportion to the frequency according to Faraday’s law. To eliminate this effect, we normalized the signals by the frequency. For 0.25 mg/ml concentration [Fig. 2(a)], the fundamental signal is the largest. The fundamental frequency (f₀) and third harmonic signals (f₃) are maximum without a DC magnetic field. As the applied DC magnetic field increases, the second harmonic signal (f₂) increases and reaches a maximum at a DC magnetic field of 6.6 mT. Contrarily, the third harmonic signal decreases with increasing DC magnetic field. The signal intensity of the second harmonic was higher than that of the third harmonic. A higher concentration of 1.25 mg/ml shows the same trend [Fig. 2(b)]. Comparing the signal peak of each harmonic signal between lower and higher concentrations, the signal at 1.25 mg/ml was five times larger than that at 0.25 mg/ml. The concentration of MNPs in this range is comparable to the iron concentrations used in previous studies of virus detection.^2,5,21–23

Figure 3 shows the number of beads (10³–10⁹ beads) dependence of the R₂/f (second harmonic signal intensity normalized by its frequency) of each MNP concentration with DC magnetic field of 6.6 mT where the second harmonic is maximum. For lower MNP concentration (0.25 mg/ml), the magnetic signal decreases with increasing the concentration of beads.²² In contrast, the magnetic signal for a higher MNP concentration (1.25 mg/ml) shows the opposite trend; the signal increases with increasing concentration. The concentration changes of magnetic nanoparticles yield the variation of the harmonic signal with different beads concentration.^21,23 In addition, the higher concentration of magnetic nanoparticles could lead the particle aggregation, indicating that the larger size of particle could increase the harmonic signal due to the larger saturation magnetic fields.²⁴ At 10³ to 10⁵ beads, the second harmonic signal intensity R₂ in the case of MNP 0.25 mg/ml changes by approximately 4%. In the case of MNP 1.25 mg/ml, R₂ changed by approximately 2% from 10³ to 10⁵ beads, indicating that the MNP 0.25 mg/ml is suitable for virus detection.

Figures 4(a) and 4(b) show the number of beads (10³ to 10⁹ beads) dependence of SNR of R₂ (the second harmonics) with DC magnetic field of 6.6 mT where the second harmonic is maximum, and R₃ (the third harmonics) without DC magnetic field in the case of MNP concentration of 0.25 mg/ml. Each data point represents the average of three measurements, and the error bars represent the standard deviation of the three measurements. For both harmonics, the SNR decreases with increasing bead concentration. The SNR of the second harmonic was larger than that of the third harmonic. In addition, the error bar of the second harmonic was smaller than that of the third harmonic. For the 10³ beads, the SNR of R₂ was 869, that of R₃ was 289, and that of R₂ was three times larger than that of R₃. In the number of beads from 10³ to 10⁵, the SNR of R₃ changed from 289 to 282, the difference in SNR was 7, and the error bars were approximately 6, which indicated that the change was equivalent to the error level. However, the SNR of R₂ changed from 869 to 831; this change was 38, and the error bars were approximately 7. This difference is much larger than the error level. We found thus that the second harmonic is superior to the third harmonic in nanobead detection, and the detectable limit is 10³ beads (∼100 pg/ml).

Figure 4(c) shows the number of beads (10³ to 10⁹ beads) dependence of D_H of each bead concentration particle in the lower concentration of 0.25 mg/ml of MNP (the particle size distributions are shown in supplementary material). The higher concentration of beads shows larger D_H of MNPs. The MNP with higher beads concentration suppress magnetic relaxation dynamics, therefore, the magnetic harmonic signals are reduced [Fig. 4(a)]. For 10³ to 10⁹ beads, D_H changes from 48.4 nm to 50.4 nm. The change of 5% in hydrodynamic size is comparable to that of a previous study² for the same level of influenza virus concentration change.

Compared with the sensitivity limit of the PCR method (∼1 pg/ml),²⁵ the sensitivity of our method (∼100 pg/ml) was inferior. However, the one-minute measurement time of our system is a strong advantage, in contrast to the several hours taken by PCR method. To improve virus detection sensitivity, we investigated the optimal AC excitation frequency, especially the lower frequency. At low frequencies, such as 10–100 Hz, there is a large change in the harmonic signal intensity with the virus concentration.²² In this case, a decrease in the SNR due to low-frequency excitation is an issue to be solved. In addition, we will apply a technique that uses three types of nanoparticles to increase D_H of MNPs by binding MNPs, the target virus, and other larger beads;²³ thus, the change in harmonic signal would possibly be larger. Besides, extending measurement time is another approach to improving detection sensitivity because the noise decreases with the square root of measurement time. However, there is a sensitivity limitation due to electromagnetic noise of the measurement devices. In terms of the detection of actual viruses, the variation of harmonic signals is expected to be larger considering the Brownian relaxation of magnetic nanoparticles based on the particle size change because the virus size (>100 nm) is larger than 60 nm beads that we employed in this study. Therefore, the same level of detection sensitivity can be expected in practical virus detections at least.

We developed a virus detection system using the second harmonics and evaluated its dependency on the DC magnetic field, MNP concentration, and nanobead concentration. For 10³ beads, the second harmonics with a larger SNR of 869 were superior to the third harmonics with an SNR of 289. In addition, we achieved a virus detection limit of 100 pg/ml using the second harmonics of MNPs in one minute. Furthermore, we confirmed that D_H of MNPs changes from 48.4 nm to 50.4 nm with increasing the concentration of beads from 10³ to 10⁹, and we found that MNP with higher bead concentration suppresses magnetic relaxation, and the magnetic second harmonic signals are reduced. The proposed method potentially enables rapid screening for virus detection.

See supplementary material for the particle size distribution of MNPs samples measured by DLS.

This work was supported by the Uehara Memorial Foundation, Hitachi Global Foundation, Nakatani Foundation, A-STEP (JPMJTR21T4), AMED (22ym0126802j0001), and the Comprehensive Growth Program for Accelerator Sciences and Joint Development Research 2022-ACCL-1 at the High Energy Accelerator Research Organization (KEK).

The authors have no conflicts to disclose.

Ryuichi Hirota: Data curation (equal); Investigation (equal); Validation (equal); Writing – original draft (lead); Writing – review & editing (equal). Toru Murayama: Resources (equal); Writing – review & editing (equal). Ryota Katsumi: Writing – review & editing (equal). Tokuhisa Kawawaki: Writing – review & editing (equal). Shin Yabukami: Writing – review & editing (equal). Ryuji Igarashi: Writing – review & editing (equal). Yuichi Negishi: Writing – review & editing (equal). Moriaki Kusakabe: Writing – review & editing (equal). Masaki Sekino: Writing – review & editing (equal). Takashi Yatsui: Writing – review & editing (equal). Akihiro kuwahata: Conceptualization (lead); Funding acquisition (lead); Investigation (lead); Supervision (lead); Writing – original draft (equal); Writing – review & editing (equal).

The data that support the findings of this study are available from the corresponding author upon reasonable request.