Person:

Lee, Hakho

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.

Low-cost, rapid and accurate detection technologies are key requisites to cope with the growing global cancer challenges. The need is particularly pronounced in resource-limited settings where treatment opportunities are often missed due to the absence of timely diagnoses. We herein describe a Holographic Assessment of Lymphoma Tissue (HALT) system that adopts a smartphone as the basis for molecular cancer diagnostics. The system detects malignant lymphoma cells labeled with marker-specific microbeads that produce unique holographic signatures. Importantly, we optimized HALT to detect lymphomas in fine-needle aspirates from superficial lymph nodes, procedures that align with the minimally invasive biopsy needs of resource-constrained regions. We equipped the platform to directly address the practical needs of employing novel technologies for “real world” use. The HALT assay generated readouts in <1.5 h and demonstrated good agreement with standard cytology and surgical pathology.

The high incidence of acute and chronic kidney injury due to various environmental factors such as heavy metals or chemicals has been a major problem in developing countries. However, the diagnosis of kidney injury in these areas can be more challenging due to the lack of highly sensitive and specific techniques that can be applied in point-of-care settings. To address this, we have developed a technique called ‘micro-urine nanoparticle detection (μUNPD)’, that allows the detection of trace amounts of molecular markers in urine. Specifically, this technique utilizes an automated on-chip assay followed by detection with a hand-held device for the read-out. Using the μUNPD technology, the kidney injury markers KIM-1 and Cystatin C were detected down to concentrations of 0.1 ng/ml and 20 ng/ml respectively, which meets the cut-off range required to identify patients with acute or chronic kidney injury. Thus, we show that the μUNPD technology enables point of care and non-invasive detection of kidney injury, and has potential for applications in diagnosing kidney injury with high sensitivity in resource-limited settings.

Monocytes are circulating macrophage and dendritic cell precursors that populate healthy and diseased tissue. In humans, monocytes consist of at least two subsets whose proportions in the blood fluctuate in response to coronary artery disease, sepsis, and viral infection. Animal studies have shown that specific shifts in the monocyte subset repertoire either exacerbate or attenuate disease, suggesting a role for monocyte subsets as biomarkers and therapeutic targets. Assays are therefore needed that can selectively and rapidly enumerate monocytes and their subsets. This study shows that two major human monocyte subsets express similar levels of the receptor for macrophage colony stimulating factor (MCSFR) but differ in their phagocytic capacity. We exploit these properties and custom-engineer magnetic nanoparticles for ex vivo sensing of monocytes and their subsets. We present a two-dimensional enumerative mathematical model that simultaneously reports number and proportion of monocyte subsets in a small volume of human blood. Using a recently described diagnostic magnetic resonance (DMR) chip with 1 (\mu)l sample size and high throughput capabilities, we then show that application of the model accurately quantifies subset fluctuations that occur in patients with atherosclerosis.

Metabolic reprogramming is widely considered a hallmark of cancer, and understanding metabolic dynamics described by the conversion rates or “fluxes” of metabolites can shed light onto biological processes of tumorigenesis and response to therapy. For real-time analysis of metabolic flux in intact cells or organisms, magnetic resonance (MR) spectroscopy and imaging methods have been developed in conjunction with hyperpolarization of nuclear spins. These approaches enable noninvasive monitoring of tumor progression and treatment efficacy and are being tested in multiple clinical trials. However, because of their limited sensitivity, these methods require a larger number of cells, on the order of 107, which is impractical for analyzing scant target cells or mass-limited samples. We present a new technology platform, a hyperpolarized micromagnetic resonance spectrometer (HMRS), that achieves real-time, 103-fold more sensitive metabolic analysis on live cells. This platform enables quantification of the metabolic flux in a wide range of cell types, including leukemia stem cells, without significant changes in viability, which allows downstream molecular analyses in tandem. It also enables rapid assessment of metabolic changes by a given drug, which may direct therapeutic choices in patients. We further advanced this platform for high-throughput analysis of hyperpolarized molecules by integrating a three-dimensionally printed microfluidic system. The HMRS platform holds promise as a sensitive method for studying metabolic dynamics in mass-limited samples, including primary cancer cells, providing novel therapeutic targets and an enhanced understanding of cellular metabolism.

Summary Despite the importance of molecular subtype classification of glioblastoma (GBM), the extent of extracellular vesicle (EV)-driven molecular and phenotypic reprogramming remains poorly understood. To reveal complex subpopulation dynamics within the heterogeneous intratumoral ecosystem, we characterized microRNA expression and secretion in phenotypically diverse subpopulations of patient-derived GBM stem-like cells (GSCs). As EVs and microRNAs convey information that rearranges the molecular landscape in a cell type-specific manner, we argue that intratumoral exchange of microRNA augments the heterogeneity of GSC that is reflected in highly heterogeneous profile of microRNA expression in GBM subtypes.

Lens-free digital in-line holography (LDIH) is a promising technology for portable, wide field-of-view imaging. Its resolution, however, is limited by the inherent pixel size of an imaging device. Here we present a new computational approach to achieve sub-pixel resolution for LDIH. The developed method is a sparsity-based reconstruction with the capability to handle the non-linear nature of LDIH. We systematically characterized the algorithm through simulation and LDIH imaging studies. The method achieved the spatial resolution down to one-third of the pixel size, while requiring only single-frame imaging without any hardware modifications. This new approach can be used as a general framework to enhance the resolution in nonlinear holographic systems.

There is currently no widely available optimal assay for diagnosing patients with enteric fever. Here we present a novel assay designed to detect amplified Salmonella nucleic acid (mRNA) using magneto-DNA probes and a miniaturized nuclear magnetic resonance device. We designed primers for genes specific to S. Typhi, S. Paratyphi A, and genes conserved among Salmonella enterica spp. and utilized strongly magnetized nanoparticles to enhance the detection signal. Blood samples spiked with in vitro grown S. Typhi, S. Paratyphi A, S. Typhimurium, and E. coli were used to confirm the specificity of each probe-set, and serial 10-fold dilutions were used to determine the limit of the detection of the assay, 0.01–1.0 CFU/ml. For proof of principle, we applied our assay to 0.5 mL blood samples from 5 patients with culture-confirmed enteric fever from Bangladesh in comparison to 3 healthy controls. We were able to detect amplified target cDNA in all 5 cases of enteric fever; no detectable signal was seen in the healthy controls. Our results suggest that a magneto-DNA nanoparticle system, with an assay time from blood collection of 3.5 hours, may be a promising platform for the rapid and culture-free diagnosis of enteric fever and non-typhoidal Salmonella bacteremia.

Real-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical problem as serial re-biopsy of primary tumours is often not a clinical option. MGMT (O6-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy. Yet, serial clinical analysis remains difficult, and, when done, primarily relies on promoter methylation studies of tumour biopsy material at the time of initial surgery. Here we present a microfluidic chip to analyse mRNA levels of MGMT and APNG in enriched tumour exosomes obtained from blood. We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients. We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.

Molecular profiling of central nervous system lymphomas in cerebrospinal fluid (CSF) samples can be challenging due to the paucicellular and limited nature of the samples. Presented herein is a microfluidic platform for complete CSF lymphoid cell analysis, including single cell capture in sub-nanoliter traps, and molecular and chemotherapeutic response profiling via on-chip imaging, all in less than one hour. The system can detect scant lymphoma cells and quantitate their kappa/lambda immunoglobulin light chain restriction patterns. The approach can be further customized for measurement of additional biomarkers, such as those for differential diagnosis of lymphoma subtypes or for prognosis, as well as for imaging exposure to experimental drugs.