Huilin Shao

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.

Ultrasensitive Clinical Enumeration of Rare Cells ex Vivo Using a Micro-Hall Detector

A hybrid microfluidic/semiconductor chip analyzes single, immunomagnetically tagged ovarian cancer cells in unprocessed biological samples. Magnetic Microchip Counts Tumor Cells The idiom “looking for a needle in a haystack” could not be applied more appropriately in medicine than to describe the detection of circulating tumor cells (CTCs). Often, only 10 of these rare cells are present in 10 ml of blood; that’s about 1 CTC for every 1 billion blood cells. Despite their scarcity, these cells may hold a wealth of information to help guide treatment decisions for cancer patients. Undaunted, Issadore and colleagues developed a miniature device that combines microfluidics and magnets to measure CTCs in patient blood at single-cell resolution. The authors designed a micro-Hall detector (μHD) that senses the magnetic moment of particles. In this system, cells were first labeled with magnetic beads conjugated to antibodies directed at a target cell surface molecule. The magnetically labeled cells could then be flowed through the microfluidic channel, where tiny Hall detectors would sense their presence. Issadore et al. first tested the μHD with cells derived from human epithelial cancers (such as breast and brain), looking for three different cancer-related markers: human epidermal growth factor receptor 2 (HER2)/neu, epidermal growth factor receptor (EGFR), and EpCAM. Out of a mixture of blood cells—some labeled, some not—the authors only missed a cancer cell 10% of the time, compared with flow cytometry (the gold standard in the clinic), which had a false-negative rate of 81%. The μHD was also able to detect multiple biomarkers on individual cells simultaneously, which could work toward further refining subpopulations of rare cells according to surface expression. To show use in the clinic, Issadore and coauthors noted elevated numbers of CTCs in the blood of 20 ovarian cancer patients, but not in any of the 15 healthy volunteers. By comparison, CellSearch (the gold standard technology for CTC enumeration) only detected CTCs in 25% of the same patient samples. The μHD appears to be a more sensitive cell counter than existing devices, with the potential to change patient management and disease monitoring in the clinic. The needles are still there, but we now have a rapid way of sorting through the haystack. The ability to detect rare cells (<100 cells/ml whole blood) and obtain quantitative measurements of specific biomarkers on single cells is increasingly important in basic biomedical research. Implementing such methodology for widespread use in the clinic, however, has been hampered by low cell density, small sample sizes, and requisite sample purification. To overcome these challenges, we have developed a microfluidic chip–based micro-Hall detector (μHD), which can directly measure single, immunomagnetically tagged cells in whole blood. The μHD can detect single cells even in the presence of vast numbers of blood cells and unbound reactants, and does not require any washing or purification steps. In addition, the high bandwidth and sensitivity of the semiconductor technology used in the μHD enables high-throughput screening (currently ~107 cells/min). The clinical use of the μHD chip was demonstrated by detecting circulating tumor cells in whole blood of 20 ovarian cancer patients at higher sensitivity than currently possible with clinical standards. Furthermore, the use of a panel of magnetic nanoparticles, distinguished with unique magnetization properties and bio-orthogonal chemistry, allowed simultaneous detection of the biomarkers epithelial cell adhesion molecule (EpCAM), human epidermal growth factor receptor 2 (HER2)/neu, and epidermal growth factor receptor (EGFR) on individual cells. This cost-effective, single-cell analytical technique is well suited to perform molecular and cellular diagnosis of rare cells in the clinic.

Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma.

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.

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]

Multicore Assemblies Potentiate Magnetic Properties of Biomagnetic Nanoparticles

Biocompatible magnetic nanoparticles (MNPs) that are both stable and amenable to chemical modification with targeting ligands continue to emerge as important materials in biomedical research.[1–5] MNPs provide an efficient contrast mechanism for biological targets; as most biological samples have intrinsically low magnetic susceptibility, only MNP-labeled objects will respond to external magnetic stimuli. Such properties have led to the widespread use of MNPs, for example, in cell sorting and bioseparation,[6, 7] medical diagnosis,[8–10] imaging,[11–14] and therapeutics.[15, 16]

Mechanism of Magnetic Relaxation Switching Sensing

Magnetic relaxation switching (MRSw) assays that employ target-induced aggregation (or disaggregation) of magnetic nanoparticles (MNPs) can be used to detect a wide range of biomolecules. The precise working mechanisms, however, remain poorly understood, often leading to confounding interpretation. We herein present a systematic and comprehensive characterization of MRSw sensing. By using different types of MNPs with varying physical properties, we analyzed the nature and transverse relaxation modes for MRSw detection. The study found that clustered MNPs are universally in a diffusion-limited fractal state (dimension of ~2.4). Importantly, a new model for transverse relaxation was constructed that accurately recapitulates observed MRSw phenomena and predicts the MRSw detection sensitivities and dynamic ranges.

Magnetic nanoparticles for biomedical NMR-based diagnostics

Summary Rapid and accurate measurements of protein biomarkers, pathogens and cells in biological samples could provide useful information for early disease diagnosis, treatment monitoring, and design of personalized medicine. In general, biological samples have only negligible magnetic susceptibility. Thus, using magnetic nanoparticles for biosensing not only enhances sensitivity but also effectively reduces sample preparation needs. This review focuses on the use of magnetic nanoparticles for in vitro detection of biomolecules and cells based on magnetic resonance effects. This detection platform, termed diagnostic magnetic resonance (DMR), exploits magnetic nanoparticles as proximity sensors, which modulate the spin–spin relaxation time of water molecules surrounding molecularly-targeted nanoparticles. By developing more effective magnetic nanoparticle biosensors, DMR detection limits for various target moieties have been considerably improved over the last few years. Already, a library of magnetic nanoparticles has been developed, in which a wide range of targets, including DNA/mRNA, proteins, small molecules/drugs, bacteria, and tumor cells, have been quantified. More recently, the capabilities of DMR technology have been further advanced with new developments such as miniaturized nuclear magnetic resonance detectors, better magnetic nanoparticles and novel conjugational methods. These developments have enabled parallel and sensitive measurements to be made from small volume samples. Thus, the DMR technology is a highly attractive platform for portable, low-cost, and efficient biomolecular detection within a biomedical setting.

Carboxymethylated Polyvinyl Alcohol Stabilizes Doped Ferrofluids for Biological Applications

[∗] Dr. M. Liong , H. Shao , Dr. J. B. Haun , Dr. H. Lee , Prof. R. Weissleder Center for Systems Biology Massachusetts General Hospital and Harvard Medical School 185 Cambridge Street, Boston, Massachusetts, 02114 (USA) E-mail: hlee@mgh.harvard.edu ; rweissleder@mgh.harvard.edu Prof. R. Weissleder Department of Systems Biology Harvard Medical School 200 Longwood Avenue, Boston, Massachusetts, 02115 (USA)

Magnetic sensing technology for molecular analyses

Magnetic biosensors, based on nanomaterials and miniature electronics, have emerged as a powerful diagnostic platform. Benefiting from the inherently negligible magnetic background of biological objects, magnetic detection is highly selective even in complex biological media. The sensing thus requires minimal sample purification and yet achieves a high signal-to-background contrast. Moreover, magnetic sensors are also well-suited for miniaturization to match the size of biological targets, which enables sensitive detection of rare cells and small amounts of molecular markers. We herein summarize recent advances in magnetic sensing technologies, with an emphasis on clinical applications in point-of-care settings. Key components of sensors, including magnetic nanomaterials, labeling strategies and magnetometry, are reviewed.