Tae-Jong Yoon

Magnetic nanoparticle biosensors

One of the major challenges in medicine is the rapid and accurate measurement of protein biomarkers, cells, and pathogens in biological samples. A number of new diagnostic platforms have recently been developed to measure biomolecules and cells with high sensitivity that could enable early disease detection or provide valuable insights into biology at the systems level. Most biological samples exhibit negligible magnetic susceptibility; therefore, magnetic nanoparticles have been used for diverse applications including biosensing, magnetic separation, and thermal ablation therapy. This review focuses on the use of magnetic nanoparticles for detection of biomolecules and cells based on magnetic resonance effects using a general detection platform termed diagnostic magnetic resonance (DMR). DMR technology encompasses numerous assay configurations and sensing principles, and to date magnetic nanoparticle biosensors have been designed to detect a wide range of targets including DNA/mRNA, proteins, enzymes, drugs, pathogens, and tumor cells. The core principle behind DMR is the use of magnetic nanoparticles as proximity sensors that modulate the spin-spin relaxation time of neighboring water molecules, which can be quantified using clinical MRI scanners or benchtop nuclear magnetic resonance (NMR) relaxometers. Recently, the capabilities of DMR technology were advanced considerably with the development of miniaturized, chip-based NMR (microNMR) detector systems that are capable of performing highly sensitive measurements on microliter sample volumes and in multiplexed format. With these and future advances in mind, DMR biosensor technology holds considerable promise to provide a high-throughput, low-cost, and portable platform for large scale molecular and cellular screening in clinical and point-of-care settings.

Magnetic nanoparticles as a catalyst vehicle for simple and easy recycling

The surface of magnetic ferrite nanoparticles (CoFe2O4) was coated with a Rh-based cationic complex, [Rh(cod)(η6-benzoic acid)]BF4, to make them homogeneously dispersable and thermodynamically stable without an excess amount of surface capping molecules in normal organic solvents. Since magnetic nanoparticles themselves have strong magnetic properties and could be considered as a nanometer-sized solid support for the surface-anchored Rh-based catalyst, the ferrite nanoparticle-supported Rh catalyst showed very effective catalytic activity toward the hydroformylation reaction of olefins and could be easily recovered from the reaction mixture by magnetic decantation to be used for subsequent reactions.

Ultrasensitive Detection of Bacteria Using Core–Shell Nanoparticles and an NMR-Filter System†

Direct detection of pathogens is key in combating human infections, in identifying nosocomial sources, in surveying food chains and in biodefense.[1] Recent advances in nanotechnology have enabled the development of new diagnostic platforms[2] aimed at more sensitive and faster pathogen detection.[3] Many of the reported technologies, albeit elegant, often fail in routine clinical settings[4] because they still require extensive specimen purification, use complex measurement setups, or are not easily scalable for clinical demands. Here we report a new, simple, nanoparticle-based platform that can rapidly detect pathogens in native biological samples. In this approach, bacteria are targeted by highly magnetic nanoparticles (MNP), concentrated into a microfluidic chamber, and detected by nuclear magnetic resonance (NMR). The clinical utility of our diagnostic platform was evaluated by detecting tuberculosis (TB), a leading cause of disease and death worldwide.[5] Using the bacillus Calmette-Guerin (BCG) as a surrogate for Mycobacterium tuberculosis, we demonstrate unprecedented detection speed and sensitivity; as few as 20 colony-forming unit (CFU) in sputum (1 mL) were detected in < 30 min. With the capability for fast, simple and portable operation, the new detection platform could be an ideal point-of-care diagnostic tool, especially in resource-limited settings. The diagnosis starts with specimen collection and incubation with bacteria specific MNP (Supporting Information Figure S1). MNP bind to the bacterial wall, rendering the bacteria superparamagnetic. In a subsequent step the spin-spin relaxation time (T2) of the whole sample is measured by NMR. As the magnetic fields from MNP dephase the precession of nuclear spins in water protons[6, 7], each MNP-tagged bacterium can shorten the T2 of billions of surrounding water molecules. To increase detection sensitivity, we have incorporated signal amplification schemes that made it possible to detect small quantities of bacteria in relatively large sample volumes. At the nanoparticle level, the detection signal has been enhanced by synthesizing Fe-based MNP with the high transverse relaxivity (r2). At the device level, the signal was amplified by concentrating bacteria into a microfluidic chamber where the NMR signal was measured. To provide portable, on-chip bacterial detection, the NMR signal was read out using a miniaturized NMR system we have recently developed.[8]. First, we developed hybrid MNP with a large Fe core and a thin ferrite shell (“cannonballs”; CB), that have very high r2 per particle (Table S1). With the limited number of binding sites per bacterium, the T2 of samples will be shorter and thereby the detection will be more sensitive when the individual MNP have higher r2. Since r2 is ∝ M2•d2, where M and d are the particle magnetization and the diameter respectively[6], we focused on making larger MNP using highly magnetic material (Fe). Figure 1a shows an example of highly mono-disperse CB (d = 16 nm). Initially, we made Fe-only MNP by thermally decomposing Fe(CO)5, followed by controlled air-oxidation to grow the ferrite shell.[9] Compared to chemical oxidation[10], our method produces a thinner shell and thereby leaves a larger Fe core (Figure S2), leading to higher M. The shell showed high crystallinity (Figure 1b) and X-ray diffraction revealed a typical pattern for a spinel structure (Figure 1c), confirming the ferrite nature of the shell.[11] The shell protected the Fe core from oxidation to maintain the magnetic properties of CB (Figure S3). CB showed high magnetization (139 emu g−1 [Fe]) and yet were superparamagnetic at room temperature (Figure 1d). Most importantly, CB assumed high r2 (= 6.1×10−11 m L s−1, 1.5 T; Figure S4), due to their high magnetization and large diameter. Figure 1 Cannonballs (CB) for bacterial targeting. a) The particles have a large metallic core (Fe) passivated with a thin ferrite shell, resulting in high particle relaxivity (> 5×10−11 mL s−1 at 0.5 T). The core diameter and the ... To render CB specific for BCG, we conjugated anti-BCG monoclonal antibodies to their surface (CB-BCG; see Methods in Supporting Information). Bacterial samples were then incubated with CB-BCG for 10 min, followed by a wash step to remove excess particles. Optical microscopy with fluorescent CB-BCG showed excellent targeting (Figure 2a). Binding of CB-BCG was further verified by the element mapping (Figure 2b), which showed high Fe signal on the bacterial membrane. The number of CB-BCG per bacterium, quantified by inductively coupled plasma atomic emission spectroscopy (ICP-AES; Figure S5), was ≈ 105. Magnetic tagging thus made the bacteria highly efficient T2-shortening agents (Figure 2c). The binding of CB-BCG to BCG could be inhibited by antibody blockade against bacterial epitopes. For control CB or when CB-BCG were used against other bacterial strains, the CB binding was minimal with T2 changes (ΔT2) < 5%. The number of CB-BCG tested against different bacteria species were < 600 per bacterium (Figure S5), confirming the specificity of CB-BCG. Figure 2 Selective BCG targeting with CB-BCG. a) Confocal micrograph of BCG incubated with fluorescently labeled CB-BCG. b) Transmission electron micrograph of BCG targeted with CB-BCG. Element mapping detected high Fe signal on a whole bacterium. c) Specificity ... To further enhance detection sensitivity and to streamline assay procedures, we next developed a chip-based filter system with NMR compatibility (Figure 3). A key component is a microfluidic chamber enclosed by a membrane filter and surrounded by a microcoil. The membrane filter performs two functions. First, it captures bacteria and concentrates them into the microfluidic chamber for NMR detection, providing a way to detect a small number of bacteria from large sample volumes. Second, the filter enables on-chip separation of bacteria from unbound CB, obviating the need for separate off-chip purification steps. Figure 4a illustrates the operating principle of the NMR-filter system. An unpurified sample is introduced into the microfluidic channel. Bacteria are retained by the membrane (pore size ≈ 100 nm), while excess CB permeate. Subsequently, a buffer solution is repeatedly injected to wash off unbound CB and the membrane is backwashed to redisperse the captured bacteria. Figure 4b shows an operation example with a sample containing BCG and CB-BCG. After sample loading, the microfluidic channel was flushed with PBS to remove unbound CB-BCG. Finally, the flow direction was reversed to resuspend the captured BCG for NMR measurements. The number of washing steps to remove unbound CB-BCG was determined by measuring T2 after each wash (Figure 4c). T2 values plateaued in 4–5 washing steps with the whole washing procedure complete in 103 times higher detection sensitivity. Figure 3 NMR-filter system for bacterial concentration and detection. a) The system consists of a microcoil and a membrane filter integrated with a microfluidic channel. The microcoil is used for NMR measurements; the membrane filter concentrates bacteria inside ... Figure 4 Bacterial separation and concentration. a) The unprocessed sample containing bacteria and CB-BCG is introduced into the device. The membrane filter retains bacteria while unbound CB-BCG pass through. To remove free CB-BCG, the microfluidic channel is ... To compare the effect of MNP relaxivity on detection sensitivity, we first used a NMR system without a membrane filter. Two types of nanoparticles, CB and cross-linked iron oxide[12] (CLIO; r2 = 7.0×10−13 mL s−1), were used (Figure 5, black and blue curves). CB-BCG showed much higher mass sensitivity, detecting as few as 6 CFU (in 1 µL sample volume), whereas the detection limit was ~100 CFU with CLIO-BCG. Using the NMR-filter system, we achieved substantially high concentration detection sensitivity (Figure 5, red curve). When BCG samples (100 µL) targeted with CB-BCG were filtered, the concentration detection limit was ~60 CFU mL−1. It is important to note that the concentration limit is theoretically unlimited with the filtering, because bacteria can be concentrated into the NMR detection chamber from large sample volumes. Figure 5 Comparison of detection sensitivity. Measurements were performed on samples with varying BCG counts to determine detection sensitivities. First, a microfluidic chip without a membrane filter was used to determine the intrinsic mass-detection limits. BCG ... To evaluate the clinical utility of the NMR-filter system, we performed comparative detection assays (Table S3). Pulmonary samples were prepared by spiking BCG into human sputa. Following liquefaction, samples were subjected to standard TB diagnostic tests, culture and acid-fast bacilli (AFB) smear microscopy, and CB-based NMR measurements. Without filtration, NMR measurements had a similar sensitivity to AFB smear microscopy, with a detection threshold of ~103 CFU/mL. However, the NMR method was less prone to human error and less labor-intensive. With the NMR-filter system, the detection sensitivity was comparable to that of culture-based detection; when 1-mL of samples were filtered, the detection limit was ~20 CFU. The NMR-based detection was much faster ( 2 weeks) and facility-dependent (e.g. incubators). These results demonstrate that the CB-based NMR-filter system can be readily applied for TB diagnosis in clinical settings.[13]

Rapid detection and profiling of cancer cells in fine-needle aspirates

There is a growing need for fast, highly sensitive and quantitative technologies to detect and profile unaltered cells in biological samples. Technologies in current clinical use are often time consuming, expensive, or require considerable sample sizes. Here, we report a diagnostic magnetic resonance (DMR) sensor that combines a miniaturized NMR probe with targeted magnetic nanoparticles for detection and molecular profiling of cancer cells. The sensor measures the transverse relaxation rate of water molecules in biological samples in which target cells of interest are labeled with magnetic nanoparticles. We achieved remarkable sensitivity improvements over our prior DMR prototypes by synthesizing new nanoparticles with higher transverse relaxivity and by optimizing assay protocols. We detected as few as 2 cancer cells in 1-μL sample volumes of unprocessed fine-needle aspirates of tumors and profiled the expression of several cellular markers in <15 min.

Trastuzumab-Conjugated Liposome-Coated Fluorescent Magnetic Nanoparticles to Target Breast Cancer

Objective To synthesize mesoporous silica-core-shell magnetic nanoparticles (MNPs) encapsulated by liposomes (Lipo [MNP@m-SiO2]) in order to enhance their stability, allow them to be used in any buffer solution, and to produce trastuzumab-conjugated (Lipo[MNP@m-SiO2]-Her2Ab) nanoparticles to be utilized in vitro for the targeting of breast cancer. Materials and Methods The physiochemical characteristics of Lipo[MNP@m-SiO2] were assessed in terms of size, morphological features, and in vitro safety. The multimodal imaging properties of the organic dye incorporated into Lipo[MNP@m-SiO2] were assessed with both in vitro fluorescence and MR imaging. The specific targeting ability of trastuzumab (Her2/neu antibody, Herceptin®)-conjugated Lipo[MNP@m-SiO2] for Her2/neu-positive breast cancer cells was also evaluated with fluorescence and MR imaging. Results We obtained uniformly-sized and evenly distributed Lipo[MNP@m-SiO2] that demonstrated biological stability, while not disrupting cell viability. Her2/neu-positive breast cancer cell targeting by trastuzumab-conjugated Lipo[MNP@m-SiO2] was observed by in vitro fluorescence and MR imaging. Conclusion Trastuzumab-conjugated Lipo[MNP@m-SiO2] is a potential treatment tool for targeted drug delivery in Her2/neu-positive breast cancer.

Body Distribution of Inhaled Fluorescent Magnetic Nanoparticles in the Mice

Body Distribution of Inhaled Fluorescent Magnetic Nanoparticles in the Mice: Jung‐Taek Kwon, et al. Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Korea—Reducing the particle size of materials is an efficient and reliable tool for improving the bioavailability of a gene or drug delivery system. In fact, nanotechnology helps in overcoming the limitations of size and can change the outlook of the world regarding science. However, a potential harmful effect of nanomaterial on workers manufacturing nanoparticles is expected in the workplace and the lack of information regarding body distribution of inhaled nanoparticles may pose serious problem. In this study, we addressed this question by studying the body distribution of inhaled nanoparticles in mice using approximately 50‐nm fluorescent magnetic nanoparticles (FMNPs) as a model of nanoparticles through nose‐only exposure chamber system developed by our group. Scanning mobility particle sizer (SMPS) analysis revealed that the mice were exposed to FMNPs with a total particle number of 4.89 × 105± 2.37 × 104/cm3(low concentration) and 9.34 × 105± 5.11 × 104/cm3(high concentration) for 4 wk (4 h/d, 5 d/wk). The body distribution of FMNPs was examined by magnetic resonance imaging (MRI) and Confocal Laser Scanning Microscope (CLSM) analysis. FMNPs were distributed in various organs, including the liver, testis, spleen, lung and brain. T2‐weighted spin‐echo MR images showed that FMNPs could penetrate the blood‐brain‐barrier (BBB). Application of nanotechnologies should not produce adverse effects on human health and the environment. To predict and prevent the potential toxicity of nanomaterials, therefore, extensive studies should be performed under different routes of exposure with different sizes and shapes of nanomaterials.

Magnetic Nanoparticles and microNMR for Diagnostic Applications

Sensitive and quantitative measurements of clinically relevant protein biomarkers, pathogens and cells in biological samples would be invaluable for disease diagnosis, monitoring of malignancy, and for evaluating therapy efficacy. Biosensing strategies using magnetic nanoparticles (MNPs) have recently received considerable attention, since they offer unique advantages over traditional detection methods. Specifically, because biological samples have negligible magnetic background, MNPs can be used to obtain highly sensitive measurements in minimally processed samples. This review focuses on the use of MNPs for in vitro detection of cellular biomarkers based on nuclear magnetic resonance (NMR) effects. This detection platform, termed diagnostic magnetic resonance (DMR), exploits MNPs as proximity sensors to modulate the spin-spin relaxation time of water molecules surrounding the molecularly-targeted nanoparticles. With new developments such as more effective MNP biosensors, advanced conjugational strategies, and highly sensitive miniaturized NMR systems, the DMR detection capabilities have been considerably improved. These developments have also enabled parallel and rapid measurements from small sample volumes and on a wide range of targets, including whole cells, proteins, DNA/mRNA, metabolites, drugs, viruses and bacteria. The DMR platform thus makes a robust and easy-to-use sensor system with broad applications in biomedicine, as well as clinical utility in point-of-care settings.

Highly Magnetic Core–Shell Nanoparticles with a Unique Magnetization Mechanism

Magnetic nanoparticles (MNPs) with high magnetic moments and very small size are under active development, since such materials have growing uses in biotechnology and medicine.[1] Ferromagnetic metals, rather than their corresponding oxides, have been suggested as an ideal constituent for MNPs for their superior magnetization.[2] Unfortunately, monometallic MNPs typically require protective layers to prevent progressive oxidation. To date, however, most core/shell approaches have yielded sub-optimal magnetization, as the shell was formed either by artificially oxidizing the core[3,4] or by coating it with non-magnetic materials.[5]

Use of Magnetic Nanoparticles to Visualize Threadlike Structures Inside Lymphatic Vessels of Rats

A novel application of fluorescent magnetic nanoparticles was made to visualize a new tissue which had not been detectable by using simple stereomicroscopes. This unfamiliar threadlike structure inside the lymphatic vessels of rats was demonstrated in vivo by injecting nanoparticles into lymph nodes and applying magnetic fields on the collecting lymph vessels so that the nanoparticles were taken up by the threadlike structures. Confocal laser scanning microscope images of cryosectioned specimens exhibited that the nanoparticles were absorbed more strongly by the threadlike structure than by the lymphatic vessels. Further examination using a transmission electron microscope revealed that the nanoparticles had been captured between the reticular fibers in the extracellular matrix of the threadlike structures. The emerging technology of nanoparticles not only allows the extremely elusive threadlike structures to be visualized but also is expected to provide a magnetically controllable means to investigate their physiological functions.

Bupivacaine Induces Apoptosis via ROS in the Schwann Cell Line

Local anesthetics have been generally accepted as being safe. However, recent clinical trials and basic studies have provided strong evidence for the neurotoxicity of local anesthetics, especially through apoptosis. We hypothesized that local anesthetics cause neural complications through Schwann cell apoptosis. Among local anesthetics tested on the Schwann cell line, RT4-D6P2T, bupivacaine significantly induced cell death, measured by the methyl tetrazolium (MTT) assay, in a dose- (LD50 = 476 μM) and time-dependent manner. The bupivacaine-induced generation of reactive oxygen species (ROS), which was initiated within 5 hrs and preceded the activation of caspase-3 and poly ADP-ribose polymerase (PARP) degradation, was suggested to trigger apoptosis, exhibited by Hoechst 33258 nuclear staining and DNA fragmentation. Furthermore, concomitant block of ROS by anti-oxidants significantly inhibited bupivacaine-induced apoptosis. Among the local anesthetics for peripheral neural blocks, bupivacaine induced apoptosis in the Schwann cell line, which may be associated with ROS production.