Nanomagnetic System for Rapid Diagnosis of Acute Infection

Abstract

Pathogen-activated antibody-secreting cells (ASCs) produce and secrete antigen-specific antibodies. ASCs are detectable in the peripheral blood as early as 3 days after antigen exposure, which makes ASCs a potential biomarker for early disease detection. Here, we present a magnetic capture and detection (MCD) assay for sensitive, on-site detection of ASCs. In this approach, ASCs are enriched through magnetic capture, and secreted antibodies are magnetically detected by a miniaturized nuclear magnetic resonance (μNMR) system. This approach is based entirely on magnetics, which supports high contrast against biological background and simplifies assay procedures. We advanced the MCD system by (i) synthesizing magnetic nanoparticles with high magnetic moments for both cell capture and antibody detection, (ii) developing a miniaturized magnetic device for high-yield cell capture, and (iii) optimizing the μNMR assay for antibody detection. Antibody responses targeting hemolysin E (HlyE) can accurately identify individuals with acute enteric fever. As a proof-of-concept, we applied MCD to detect antibodies produced by HlyE-specific hybridoma cells. The MCD achieved high sensitivity in detecting antibodies secreted from as few as 5 hybridoma cells (50 cells/mL). Importantly, the assay could be performed with whole blood with minimal sample processing.

Keywords: acute infections; biosensors; enteric fever; host response; magnetic nanoparticles; nuclear magnetic resonance.

Figure 1.. Magnetic particles for the MCD assay.

(A) Transmission electron microscope (TEM) image of zinc (Zn) ferrite MNPs (Zn_0.4Fe_2.6O₄). The MNPs have an average diameter of ~13 nm and have higher magnetization than other ferrite types.^, (B) To use the Zn ferrite MNPs as a μNMR sensing agent, the MNPs were coated with dextran and further functionalized with streptavidin (StAv). (C) Comparison of transverse relaxivity (r₂) of MNPs. The Zn ferrite MNPs show the highest r₂ value owing to their high magnetic moment. CLIO, cross linked iron oxides. (D) The scanning electron microscope (SEM) image of cell capture Zn ferrite beads (that is, Zn ferrite MNPs-embedded polystyrene microbeads). Enlarged pseudo color-mapped image (right panel) show presence of the Zn ferrite MNPs (yellow). (E) The Zn ferrite beads show higher saturation magnetization (115 emu/g [metal]) than conventional magnetic beads with maghemite cores (83 emu/g [metal]).

Figure 2.. Checkerboard magnetic chip for cell capture.

(A) The checkerboard array consists of small magnets with each magnet having an alternating polarity. A pair of arrays are used. (B) Magnetic field strength (B_z) between two checkerboard arrays. The field is tightly bound to the magnet surface, creating a high field gradient. (C) The checkerboard arrays generate a larger magnetic force compared to a simple two-pole system. The force is simulated for magnetic beads with 1 μm radius and unit susceptibility. (D) A cell separator was constructed by placing a fluidic device between checkerboard arrays. (E) Mixtures of magnetic and non-magnetic particles were processed at different flow rates, and the enrichment ratio was measured. The system achieved high enrichment ratio (~3,500) even at 20 mL/hr flow rate. (F) Magnetic enrichment of hybridoma cells. Cells were labeled with CD45-specific magnetic beads; IgG2b-beads were used as a control. The recovery rate was >95% for targeted samples. The flow rate was 2 mL/hr.

Figure 3.. Magneto-antibody assay.

(A) Detection sensitivity for antibody detection. Anti-HlyE monoclonal antibodies were captured on polystyrene microbeads conjugated with HlyE. Captured antibodies were coupled with Zn ferrite MNPs via secondary antibodies, and the samples were measured by μNMR. Titration experiments established the limit of detection (LOD) of 59 pg/mL (~1.7 pM). (B) When compared to ELISA, the magneto-antibody assay showed an excellent match (R² > 0.99). a.u., arbitrary unit. (C) Detection specificity was examined. We varied the capture antigen on the beads (hemolysin E/HlyE, keyhole limpet hemocyanin/KLH) as well as target antibodies (anti-HlyE, anti-TT/tetanus toxoid). Only the matching pair (HlyE-bead and anti-HlyE antibody) led to high signal. (D, E) Detection dynamics with respect to culture time (D) and hybridoma cell numbers (E) was measured. The threshold was set at 3 × standard deviation (s.d.) above background signal of the sample without target antibodies. (F) A generalized detection curve was constructed using data from (D, E). For clinical ASC ranges (50 ~ 1000 cells), the required culture time was about 1 hour. The dotted line indicates detection limit with Zn ferrite MNPs, and the white dots are measured LODs.

Figure 4.. Performance of MCD using spiked blood samples.

(A) The effect of magnetic selection on antibody secretion was tested. Hybridoma cells were labeled with CD45-specific magnetic beads and processed by the checkerboard magnetic chip. Captured cells were on-chip cultured, and secreted antibodies were measured by μNMR. No significant difference (P > 0.5; t-test) between positive control and CD45-captured samples was observed, while these samples were easily distinguished from negative control. (B) Micrograph of hybridoma cells captured by anti-CD45 magnetic beads and stained by DRAQ5 nucleus staining dyes (1:250 dilution). (C) Detection of HlyE-specific antibodies from cells captured from spiked whole blood. The high ΔR₂ is obtained only with the matching pair (HlyE-specific hybridoma cells and HlyE-bead). (D) Detection of antibodies in clinical samples. Patient samples with blood culture-confirmed enteric fever were collected from a typhoid/paratyphoid endemic region (Dhaka, Bangladesh). Healthy controls were from the US and Bangladesh. Overall, patient samples showed higher level of HlyE-specific antibodies.

Scheme 1.. Detection strategy.

Antibody secreting cells (ASCs) transiently migrate in the peripheral blood peaking 7 days after infection. In the MCD (magnetic capture and detection) approach, ASCs are enriched through magnetic capture, and briefly cultured on-chip to increase antibody concentrations. Secreted antigen-specific antibodies are then detected by micro nuclear magnetic resonance (μNMR). Magnetic nanoparticles (MNPs) with high magnetic moments are used i) as a core material for magnetic beads (MBs) in cell separation, and ii) as a μNMR sensing agent.