Hakho Lee

Chip-NMR biosensor for detection and molecular analysis of cells

Rapid and accurate measurement of biomarkers in tissue and fluid samples is a major challenge in medicine. Here we report the development of a new, miniaturized diagnostic magnetic resonance (DMR) system for multiplexed, quantitative and rapid analysis. By using magnetic particles as a proximity sensor to amplify molecular interactions, the handheld DMR system can perform measurements on unprocessed biological samples. We show the capability of the DMR system by using it to detect bacteria with high sensitivity, identify small numbers of cells and analyze them on a molecular level in real time, and measure a series of protein biomarkers in parallel. The DMR technology shows promise as a robust and portable diagnostic device.

Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor

Exosomes show potential for cancer diagnostics because they transport molecular contents of the cells from which they originate. Detection and molecular profiling of exosomes is technically challenging and often requires extensive sample purification and labeling. Here we describe a label-free, high-throughput approach for quantitative analysis of exosomes. Our nano-plasmonic exosome (nPLEX) assay is based on transmission surface plasmon resonance through periodic nanohole arrays. Each array is functionalized with antibodies to enable profiling of exosome surface proteins and proteins present in exosome lysates. We show that this approach offers improved sensitivity over previous methods, enables portable operation when integrated with miniaturized optics and allows retrieval of exosomes for further study. Using nPLEX to analyze ascites samples from ovarian cancer patients, we find that exosomes derived from ovarian cancer cells can be identified by their expression of CD24 and EpCAM, suggesting the potential of exosomes for diagnostics.

Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis

Genetic screens are powerful tools for identifying genes responsible for diverse phenotypes. Here we describe a genome-wide CRISPR/Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library with 67,405 single-guide RNAs (sgRNAs). The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late-stage primary tumors were found to target a small set of genes, suggesting that specific loss-of-function mutations drive tumor growth and metastasis. Individual sgRNAs and a small pool of 624 sgRNAs targeting the top-scoring genes from the primary screen dramatically accelerate metastasis. In all of these experiments, the effect of mutations on primary tumor growth positively correlates with the development of metastases. Our study demonstrates Cas9-based screening as a robust method to systematically assay gene phenotypes in cancer evolution in vivo.

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.

Microelectromagnets for the control of magnetic nanoparticles

A microelectromagnet matrix and a ring trap that position and control magnetic nanoparticles are demonstrated. They consist of multiple layers of lithographically defined Au wires separated by transparent, insulating polyimide layers on sapphire substrates. Magnetic fieldpatterns produced by these devices allow microscopically precise control and manipulation of magnetic nanoparticles. A microelectromagnet matrix produces single or multiple peaks in the magnetic field magnitude, which trap, move, and rotate magnetic nanoparticles, as well as electromagnetic fields to probe and detect particles. Microelectromagnets are new tools with which to study and manipulate nanoparticles and biological entities.

Manipulation of biological cells using a microelectromagnet matrix

Noninvasive manipulation of biological cells inside a microfluidic channel was demonstrated using a microelectromagnet matrix. The matrix consists of two layers of straight Au wires, aligned perpendicular to each other, that are covered by insulating layers. By adjusting the current in each independent wire, the microelectromagnet matrix can create versatile magnetic field patterns to control the motion of individual cells in fluid. Single or multiple yeast cells attached to magnetic beads were trapped, continuously moved and rotated, and a viable cell was separated from nonviable cells for cell sorting.

Bioorthogonal chemistry amplifies nanoparticle binding and enhances the sensitivity of cell detection

Nanoparticles have emerged as key materials for biomedical applications because of their unique and tunable physical properties, multivalent targeting capability, and high cargo capacity. Motivated by these properties and by current clinical needs, numerous diagnostic and therapeutic nanomaterials have recently emerged. Here we describe a novel nanoparticle targeting platform that uses a rapid, catalyst-free cycloaddition as the coupling mechanism. Antibodies against biomarkers of interest were modified with trans-cyclooctene and used as scaffolds to couple tetrazine-modified nanoparticles onto live cells. We show that the technique is fast, chemoselective, adaptable to metal nanomaterials, and scalable for biomedical use. This method also supports amplification of biomarker signals, making it superior to alternative targeting techniques including avidin/biotin.

Incorporation of Iron Oxide Nanoparticles and Quantum Dots into Silica Microspheres

We describe the synthesis of magnetic and fluorescent silica microspheres fabricated by incorporating maghemite (gamma-Fe2O3) nanoparticles (MPs) and CdSe/CdZnS core/shell quantum dots (QDs) into a silica shell around preformed silica microspheres. The resultant approximately 500 nm microspheres have a narrow size distribution and show uniform incorporation of QDs and MPs into the shell. We have demonstrated manipulation of these microspheres using an external magnetic field with real-time fluorescence microscopy imaging.

A magneto-DNA nanoparticle system for rapid detection and phenotyping of bacteria

So far, although various diagnostic approaches for pathogen detection have been proposed, most are too expensive, lengthy or limited in specificity for clinical use. Nanoparticle systems with unique material properties, however, circumvent these problems and offer improved accuracy over current methods. Here, we present novel magneto-DNA probes capable of rapid and specific profiling of pathogens directly in clinical samples. A nanoparticle hybridization assay, involving ubiquitous and specific probes that target bacterial 16S rRNAs, was designed to detect amplified target DNAs using a miniaturized NMR device. Ultimately, the magneto-DNA platform will allow both universal and specific detection of various clinically relevant bacterial species, with sensitivity down to single bacteria. Furthermore, the assay is robust and rapid, simultaneously diagnosing a panel of 13 bacterial species in clinical specimens within 2 h. The generic platform described could be used to rapidly identify and phenotype pathogens for a variety of applications.

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]