David Issadore

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.

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.

Ubiquitous Detection of Gram-Positive Bacteria with Bioorthogonal Magnetofluorescent Nanoparticles

The ability to rapidly diagnose gram-positive pathogenic bacteria would have far reaching biomedical and technological applications. Here we describe the bioorthogonal modification of small molecule antibiotics (vancomycin and daptomycin), which bind to the cell wall of gram-positive bacteria. The bound antibiotics conjugates can be reacted orthogonally with tetrazine-modified nanoparticles, via an almost instantaneous cycloaddition, which subsequently renders the bacteria detectable by optical or magnetic sensing. We show that this approach is specific, selective, fast and biocompatible. Furthermore, it can be adapted to the detection of intracellular pathogens. Importantly, this strategy enables detection of entire classes of bacteria, a feat that is difficult to achieve using current antibody approaches. Compared to covalent nanoparticle conjugates, our bioorthogonal method demonstrated 1-2 orders of magnitude greater sensitivity. This bioorthogonal labeling method could ultimately be applied to a variety of other small molecules with specificity for infectious pathogens, enabling their detection and diagnosis.

Specific pathogen detection using bioorthogonal chemistry and diagnostic magnetic resonance.

The development of faster and more sensitive detection methods capable of identifying specific bacterial species and strains has remained a longstanding clinical challenge. Thus to date, the diagnosis of bacterial infections continues to rely on the performance of time-consuming microbiological cultures. Here, we demonstrate the use of bioorthogonal chemistry for magnetically labeling specific pathogens to enable their subsequent detection by nuclear magnetic resonance. Antibodies against a bacterial target of interest were first modified with trans-cyclooctene and then coupled to tetrazine-modified magnetic nanoprobes, directly on the bacteria. This labeling method was verified by surface plasmon resonance as well as by highly specific detection of Staphylococcus aureus using a miniaturized diagnostic magnetic resonance system. Compared to other copper-free bioorthogonal chemistries, the cycloaddition reaction reported here displayed faster kinetics and yielded higher labeling efficiency. Considering the short assay times and the portability of the necessary instrumentation, it is feasible that this approach could be adapted for clinical use in resource-limited settings.

Rare cell isolation and profiling on a hybrid magnetic/size-sorting chip

We present a hybrid magnetic/size-sorting (HMSS) chip for isolation and molecular analyses of circulating tumor cells (CTCs). The chip employs both negative and positive cell selection in order to provide high throughput, unbiased CTC enrichment. Specifically, the system utilizes a self-assembled magnet to generate high magnetic forces and a weir-style structure for cell sorting. The resulting device thus can perform multiple functions, including magnetic depletion, size-selective cell capture, and on-chip molecular staining. With such capacities, the HMSS device allowed one-step CTC isolation and single cell detection from whole blood, tested with spiked cancer cells. The system further facilitated the study of individual CTCs for heterogeneity in molecular marker expression.

Microchip-based detection of magnetically labeled cancer biomarkers.

Micro-magnetic sensing and actuation have emerged as powerful tools for the diagnosis and monitoring of cancer. These technologies can be miniaturized and integrated onto compact, microfluidic platforms, enabling molecular diagnostics to be performed in practical clinical settings. Molecular targets tagged with magnetic nanoparticles can be detected with high sensitivity directly in unprocessed clinical samples (e.g. blood, sputum) due to the inherently negligible magnetic susceptibility of biological material. As a result, magnetic microchip-based diagnostics have been applied with great success to the isolation and detection of rare cells and the measurement of sparse soluble proteins. In this paper, we review recent advances in microchip-based detection of magnetically labeled biomarkers and their translation to clinical applications in cancer.

Point-of-care diagnostics on a chip /

Point-of-care diagnostics on a chip / , Point-of-care diagnostics on a chip / , کتابخانه دیجیتال جندی شاپور اهواز

Integrated Circuit / Microfluidic Chip for Programmable Cell and Droplet Manipulation with Dielectrophoresis

We present an integrated circuit/microfluidic chip that traps and moves individual living biological cells and chemical droplets along programmable paths using dielectrophoresis (DEP). Our chip combines the biocompatibility of microfluidics with the programmability and complexity of integrated circuits (ICs). The chip is capable of simultaneously and independently controlling the location of thousands of dielectric objects, such as cells and chemical droplets. The chip consists of an array of 128 x 256 pixels, 11 x 11 microm(2) in size, controlled by built-in SRAM memory; each pixel can be energized by a radio frequency (RF) voltage of up to 5 V(pp). The IC was built in a commercial foundry and the microfluidic chamber was fabricated on its top surface at Harvard. Using this hybrid chip, we have moved yeast and mammalian cells through a microfluidic chamber at speeds up to 30 microm sec(-1). Thousands of cells can be individually trapped and simultaneously positioned in controlled patterns. The chip can trap and move pL droplets of water in oil, split one droplet into two, and mix two droplets into one. Our IC/microfluidic chip provides a versatile platform to trap and move large numbers of cells and fluid droplets individually for lab-on-a-chip applications.

Smartphone-enabled optofluidic exosome diagnostic for concussion recovery

A major impediment to improving the treatment of concussion is our current inability to identify patients that will experience persistent problems after the injury. Recently, brain-derived exosomes, which cross the blood-brain barrier and circulate following injury, have shown great potential as a noninvasive biomarker of brain recovery. However, clinical use of exosomes has been constrained by their small size (30–100 nm) and the extensive sample preparation (>24 hr) needed for traditional exosome measurements. To address these challenges, we developed a smartphone-enabled optofluidic platform to measure brain-derived exosomes. Sample-to-answer on our chip is 1 hour, 10x faster than conventional techniques. The key innovation is an optofluidic device that can detect enzyme amplified exosome biomarkers, and is read out using a smartphone camera. Using this approach, we detected and profiled GluR2+ exosomes in the post-injury state using both in vitro and murine models of concussion.

Nanoparticle Mediated Measurement of Target-Drug Binding in Cancer Cells

Responses to molecularly targeted therapies can be highly variable and depend on mutations, fluctuations in target protein levels in individual cells, and drug delivery. The ability to rapidly quantitate drug response in cells harvested from patients in a point-of-care setting would have far reaching implications. Capitalizing on recent developments with miniaturized NMR technologies, we have developed a magnetic nanoparticle-based approach to directly measure both target expression and drug binding in scant human cells. The method involves covalent conjugation of the small-molecule drug to a magnetic nanoparticle that is then used as a read-out for target expression and drug-binding affinity. Using poly(ADP-ribose) polymerase (PARP) inhibition as a model system, we developed an approach to distinguish differential expression of PARP in scant cells with excellent correlation to gold standards, the ability to mimic drug pharmacodynamics ex vivo through competitive target-drug binding, and the potential to perform such measurements in clinical samples.