Page 19 - Read Online

Page 14 of 21 Su et al. J Cancer Metastasis Treat 2020;6:19 I http://dx.doi.org/10.20517/2394-4722.2020.48

imaging (MRI) [83-87] . Since the biological samples show negligible magnetic susceptibility, MNPs are
introduced as contrast agents for in vitro detection of biomolecules and cells based on NMR in recent
years [88-91] . For most NMR-based applications, the primary NMR signals come from hydrogen nuclei
contained within water or lipid molecules, due to: (1) intrinsic nonzero nuclear spins; and (2) natural
abundance in the form of water and lipid molecules.

The foregoing sections describe MNP-based bioassays using technologies such as magnetoresistive and
Hall sensors that directly measure the magnetic fields from MNP-labeled biological compounds. However,
for MNP enhanced NMR bioassays, NMR spectroscopy exploits MNPs as proximity sensors that modulate
the spin-spin relaxation time of water molecules adjacent to the MNPs, where the MNPs generate local
dipolar fields that modulate the proton relaxation rates of billions of neighboring water molecules; thus, the
analytical signals are directly generated from the whole sample volume. These MNPs are contrast agents in
NMR to improve sensitivity in turbid samples with reduced sample preparation. In this section, we review
the NMR-based diagnostics using MNPs for a wide range of applications including DNA, RNA, proteins,
small molecules, tumor cells, etc.

NMR-based cancer diagnostics
MNPs along with NMR has been applied for many in vitro liquid biopsies including the detection of
Listeria monocytogenes, Salmonella, and Cronobacter sakazakii from food [92,93] as well as the microcystin-
[94]
LR (MC-LR) from water . In this section, we review the applications in the area of cancer detection.
Lee’s group reported a DMR (diagnostic magnetic resonance) system based on NMR technology and
MNP contrast agents. Conceptually, the DMR system consists of a microNMR (μNMR) chip containing
microcoils, a microfluidic network for sample handling, on-board NMR electronics, and a small permanent
magnet, as shown in Figure 9B-D. The microfluidic network in Figure 9C can effectively mix MNPs and
biofluid sample (thus speeding up the specific binding to target analytes) by generating chaotic advection
through the meandering channels. The NMR circuits in Figure 9E are designed to perform T2 and T1
measurements. The detection of MNP-labeled tumor cells is achieved by exploiting the T2-shortening
effect of MNPs in NMR measurements, where the MNPs generate local dipolar fields with strong spatial
dependence to destroy the coherence in the T2 relaxation of neighboring water protons. These MNP-
labeled cells consequently show faster decay of NMR signal, or shorter T2 relaxation time than non-
targeted cells, as shown in Figure 9A.

[95]
Using this DMR system, Ghazani et al. reported the detection of circulating tumor cells (CTCs) directly
from whole blood sample without primary purification. The detection is achieved by the combined and
simultaneous sensing of four cancer biomarkers: EpCAM, HER-2, EGFR, and MUC-1. The monoclonal
antibodies against EpCAM, HER-2, EGFR, and MUC-1 are conjugated with (E)-cyclooct-4-enyl
2,5-dioxopyrrolidin-1-yl carbonate (TCO-NHS), while the MNPs are tetrazine (Tz) modified. Each whole
blood sample (7 mL) is lysed and cell pellets resuspended in PBS buffer. Then, TCO-modified antibodies
are added and incubated for 15 min. Samples are washed twice and tetrazine-modified MNPs are added
and incubated for 15 min. After another two cycles of washing steps, the mixture is suspended in 20 μL of
PBS for μNMR measurements. The total labeling and incubation procedure takes approximately 30 min,
as shown in Figure 10A. They reported the NMR measurements on 58 patients with confirmed epithelial
malignancies. As shown in Figure 10B, these blood samples from 58 patients were profiled for four cancer
biomarkers: EpCAM, MUC-1, HER-2, and EGFR. Although a notable fraction of the samples shows
negative results for each biomarker, the combined biomarker analysis is able to correctly identify 99.2% of
samples as malignant. As a comparison, Khosravi et al. [96,97] reported a nanotube-antibody micro-array chip
for the detection of breast cancer cells from 0.85 mL spiked blood. A ∼90% sensitivity and a 90% specificity
in capture of 1000 SKBR3 breast cancer cells in blood using anti-Her2-functionalized devices are achieved.