Automated Image Analysis for Characterization of Circulating Tumor Cells and Clusters Sorted by Magnetic Levitation.

Magnetic levitation-based sorting technologies have revolutionized the detection and isolation of rare cells, including circulating tumor cells (CTCs) and circulating tumor cell clusters (CTCCs). Manual counting and quantification of these cells are prone to time-consuming processes, human error, and inter-observer variability, particularly challenging when heterogeneous cell types in 3D clusters are present. To overcome these challenges, we developed "Fastcount," an in-house MATLAB-based algorithm for precise, automated quantification and phenotypic characterization of CTCs and CTCCs, in both 2D and 3D. Fastcount is 120 times faster than manual counting and produces reliable results with a ±7.3% deviation compared to a trained laboratory technician. By analyzing 400 GB of fluorescence imaging data, we showed that Fastcount outperforms manual counting and commercial software when cells are aggregated in 3D or staining artifacts are present, delivering more accurate results. We further employed Fastcount for automated analysis of 3D image stacks obtained from CTCCs isolated from colorectal adenocarcinoma and renal cell carcinoma blood samples. Interestingly, we observed a highly heterogeneous spatial cellular composition within CTCCs, even among clusters from the same patient. Overall, Fastcount can be employed for various applications with lab-chip devices, such as CTC detection, CTCC analysis in 3D and cell detection in biosensors.

Size and density measurements of single sickle red blood cells using microfluidic magnetic levitation.

Single cells have unique biophysical signatures that can rapidly change during various disease states. For instance, cellular density is an inherent property differing between cell types. Characterizing changes in fundamental density properties down to the single-cell level can reveal sub-populations in pathological states. Here, we have developed a microfluidic, magnetic levitation-based assay (MagDense) that detects minute density differences of individual red blood cells (RBCs) down to 0.0001 g mL-1 resolution. This assay fractionates RBCs based on their density profiles in a non-ionic paramagnetic medium flowing in a capillary microchannel placed between magnets with same poles facing each other. Based on precisely measured levitation height and density of individual RBCs at their specific equilibrium state, we demonstrated that MagDense can accurately analyze the density of sickle hemoglobin (HbS)-containing RBCs and normal hemoglobin (HbA)-containing RBCs. In addition, the precise density and cell size measurements at the single cell level showed three different sub-populations of RBCs in blood samples from individuals with homozygous sickle cell disease receiving blood transfusions; where less dense, HbA-containing RBCs levitated higher, while the denser, HbS-containing RBCs levitated lower. We compared the mean RBC densities of sickle cell disease subjects with healthy controls and found distinctly separated bands of RBC density for each group denoting the likely range of cell densities seen in the blood samples. The high resolution of our method enabled measurement of deviation from the mean RBC density. Moreover, we introduced a new term as a measure of density dispersion, "RBC levitational density width, RLDW". Mean RBC density in sickle cell disease associated with hemoglobin from complete blood count (p = 0.032, linear regression) and RLDW associated with absolute reticulocyte count (ARC) and RBC distribution width (RDW) from complete blood count (p = 0.002 for ARC and p = 003 for RDW, linear regression). Our magnetic levitation-based assay enables rapid, accurate, density-based imaging, profiling and label-free monitoring of single RBCs. Our approach can be broadly applicable to investigate blood cell disorders and the effects of emerging pharmacological and curative therapies in patient outcomes.

Multiparametric biophysical profiling of red blood cells in malaria infection.

Biophysical separation promises label-free, less-invasive methods to manipulate the diverse properties of live cells, such as density, magnetic susceptibility, and morphological characteristics. However, some cellular changes are so minute that they are undetectable by current methods. We developed a multiparametric cell-separation approach to profile cells with simultaneously changing density and magnetic susceptibility. We demonstrated this approach with the natural biophysical phenomenon of Plasmodium falciparum infection, which modifies its host erythrocyte by simultaneously decreasing density and increasing magnetic susceptibility. Current approaches have used these properties separately to isolate later-stage infected cells, but not in combination. We present biophysical separation of infected erythrocytes by balancing gravitational and magnetic forces to differentiate infected cell stages, including early stages for the first time, using magnetic levitation. We quantified height distributions of erythrocyte populations-27 ring-stage synchronized samples and 35 uninfected controls-and quantified their unique biophysical signatures. This platform can thus enable multidimensional biophysical measurements on unique cell types.

Levitational Cell Cytometry for Forensics.

Here, a method for label-free, real-time interrogation, monitoring, detection, and sorting of biological rare cells in magnetically suspended heterogeneous samples is developed. To achieve this, heterogeneous populations of cells are levitated and confined in a microcapillary channel. This strategy enables spatiotemporal differential magnetic levitation of rare fragile dead cells equilibrating at different heights based on the balance between magnetic and corrected gravitational forces. In addition, the sorting of fragile rare dead cell populations is monitored in real-time. This technique provides a broadly applicable label-free tool for high resolution, real-time research, as well as forensic evidence processing of rape kits. This method is validated with forensic mock samples dating back to 2003, isolating sperm from epithelial cells (E. cells) with >90% efficiency and >97% purity. Overall, this method reduces the processing time by over 20-fold down to 20 min, eliminating centrifugation and labels, and providing an inexpensive and high-yield alternative to the current centrifuge-based differential extraction techniques. It can potentially facilitate the forensic downstream genomic analyses, accelerating the identification of suspects, and advancing public safety.

Levitating Cells to Sort the Fit and the Fat.

Density is a core material property and varies between different cell types, mainly based on differences in their lipid content. Sorting based on density enables various biomedical applications such as multi-omics in precision medicine and regenerative repair in medicine. However, a significant challenge is sorting cells of the same type based on density differences. Here, a new method for real-time monitoring and sorting of single cells based on their inherent levitation profiles driven by their lipid content is reported. As a model system, human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) from a patient with neutral lipid storage disease (NLSD) due to loss of function of adipose triglyceride lipase (ATGL) resulting in abnormal lipid storage in cardiac muscle are used. This levitation-based strategy detects subpopulations within ATGL-deficient hiPSC-CMs with heterogenous lipid content, equilibrating at different levitation heights due to small density differences. In addition, sorting of these differentially levitating subpopulations are monitored in real time. Using this approach, sorted healthy and diseased hiPSC-CMs maintain viability and function. Pixel-tracking technologies show differences in contraction between NLSD and healthy hiPSC-CMs. Overall, this is a unique approach to separate diseased cell populations based on their intracellular lipid content that cannot be achieved using traditional flow cytometry techniques.

A Novel On-Chip Method for Differential Extraction of Sperm in Forensic Cases.

One out of every six American women has been the victim of a sexual assault in their lifetime. However, the DNA casework backlog continues to increase outpacing the nation's capacity since DNA evidence processing in sexual assault casework remains a bottleneck due to laborious and time-consuming differential extraction of victim's and perpetrator's cells. Additionally, a significant amount (60-90%) of male DNA evidence may be lost with existing procedures. Here, a microfluidic method is developed that selectively captures sperm using a unique oligosaccharide sequence (Sialyl-LewisX), a major carbohydrate ligand for sperm-egg binding. This method is validated with forensic mock samples dating back to 2003, resulting in 70-92% sperm capture efficiency and a 60-92% reduction in epithelial fraction. Captured sperm are then lysed on-chip and sperm DNA is isolated. This method reduces assay-time from 8 h to 80 min, providing an inexpensive alternative to current differential extraction techniques, accelerating identification of suspects and advancing public safety.

Magnetically Guided Self-Assembly and Coding of 3D Living Architectures.

In nature, cells self-assemble at the microscale into complex functional configurations. This mechanism is increasingly exploited to assemble biofidelic biological systems in vitro. However, precise coding of 3D multicellular living materials is challenging due to their architectural complexity and spatiotemporal heterogeneity. Therefore, there is an unmet need for an effective assembly method with deterministic control on the biomanufacturing of functional living systems, which can be used to model physiological and pathological behavior. Here, a universal system is presented for 3D assembly and coding of cells into complex living architectures. In this system, a gadolinium-based nonionic paramagnetic agent is used in conjunction with magnetic fields to levitate and assemble cells. Thus, living materials are fabricated with controlled geometry and organization and imaged in situ in real time, preserving viability and functional properties. The developed method provides an innovative direction to monitor and guide the reconfigurability of living materials temporally and spatially in 3D, which can enable the study of transient biological mechanisms. This platform offers broad applications in numerous fields, such as 3D bioprinting and bottom-up tissue engineering, as well as drug discovery, developmental biology, neuroscience, and cancer research.

Multifunctional, inexpensive, and reusable nanoparticle-printed biochip for cell manipulation and diagnosis.

Isolation and characterization of rare cells and molecules from a heterogeneous population is of critical importance in diagnosis of common lethal diseases such as malaria, tuberculosis, HIV, and cancer. For the developing world, point-of-care (POC) diagnostics design must account for limited funds, modest public health infrastructure, and low power availability. To address these challenges, here we integrate microfluidics, electronics, and inkjet printing to build an ultra-low-cost, rapid, and miniaturized lab-on-a-chip (LOC) platform. This platform can perform label-free and rapid single-cell capture, efficient cellular manipulation, rare-cell isolation, selective analytical separation of biological species, sorting, concentration, positioning, enumeration, and characterization. The miniaturized format allows for small sample and reagent volumes. By keeping the electronics separate from microfluidic chips, the former can be reused and device lifetime is extended. Perhaps most notably, the device manufacturing is significantly less expensive, time-consuming, and complex than traditional LOC platforms, requiring only an inkjet printer rather than skilled personnel and clean-room facilities. Production only takes 20 min (vs. up to weeks) and $0.01-an unprecedented cost in clinical diagnostics. The platform works based on intrinsic physical characteristics of biomolecules (e.g., size and polarizability). We demonstrate biomedical applications and verify cell viability in our platform, whose multiplexing and integration of numerous steps and external analyses enhance its application in the clinic, including by nonspecialists. Through its massive cost reduction and usability we anticipate that our platform will enable greater access to diagnostic facilities in developed countries as well as POC diagnostics in resource-poor and developing countries.

Monitoring Neutropenia for Cancer Patients at the Point of Care.

Neutrophils have a critical role in regulating the immune system. The immune system is compromised during chemotherapy, increasing infection risks and imposing a need for regular monitoring of neutrophil counts. Although commercial hematology analyzers are currently used in clinical practice for neutrophil counts, they are only available in clinics and hospitals, use large blood volumes, and are not available at the point of care (POC). Additionally, phlebotomy and blood processing require trained personnel, where patients are often admitted to hospitals when the infections are at late stage due to lack of frequent monitoring. Here, a reliable method is presented that selectively captures and quantifies white blood cells (WBCs) and neutrophils from a finger prick volume of whole blood by integrating microfluidics with high-resolution imaging algorithms. The platform is compact, portable, and easy to use. It captures and quantifies WBCs and neutrophils with high efficiency (>95%) and specificity (>95%) with an overall 4.2% bias compared to standard testing. The results from a small cohort of patients (N = 11 healthy, N = 5 lung and kidney cancer) present a unique disposable cell counter, demonstrating the ability of this tool to monitor neutrophil and WBC counts within clinical or in resource-constrained environments.

Integrating Cell Phone Imaging with Magnetic Levitation (i-LEV) for Label-Free Blood Analysis at the Point-of-Living.

There is an emerging need for portable, robust, inexpensive, and easy-to-use disease diagnosis and prognosis monitoring platforms to share health information at the point-of-living, including clinical and home settings. Recent advances in digital health technologies have improved early diagnosis, drug treatment, and personalized medicine. Smartphones with high-resolution cameras and high data processing power enable intriguing biomedical applications when integrated with diagnostic devices. Further, these devices have immense potential to contribute to public health in resource-limited settings where there is a particular need for portable, rapid, label-free, easy-to-use, and affordable biomedical devices to diagnose and continuously monitor patients for precision medicine, especially those suffering from rare diseases, such as sickle cell anemia, thalassemia, and chronic fatigue syndrome. Here, a magnetic levitation-based diagnosis system is presented in which different cell types (i.e., white and red blood cells) are levitated in a magnetic gradient and separated due to their unique densities. Moreover, an easy-to-use, smartphone incorporated levitation system for cell analysis is introduced. Using our portable imaging magnetic levitation (i-LEV) system, it is shown that white and red blood cells can be identified and cell numbers can be quantified without using any labels. In addition, cells levitated in i-LEV can be distinguished at single-cell resolution, potentially enabling diagnosis and monitoring, as well as clinical and research applications.

Magnetic levitation of single cells.

Several cellular events cause permanent or transient changes in inherent magnetic and density properties of cells. Characterizing these changes in cell populations is crucial to understand cellular heterogeneity in cancer, immune response, infectious diseases, drug resistance, and evolution. Although magnetic levitation has previously been used for macroscale objects, its use in life sciences has been hindered by the inability to levitate microscale objects and by the toxicity of metal salts previously applied for levitation. Here, we use magnetic levitation principles for biological characterization and monitoring of cells and cellular events. We demonstrate that each cell type (i.e., cancer, blood, bacteria, and yeast) has a characteristic levitation profile, which we distinguish at an unprecedented resolution of 1 × 10(-4) g ⋅ mL(-1). We have identified unique differences in levitation and density blueprints between breast, esophageal, colorectal, and nonsmall cell lung cancer cell lines, as well as heterogeneity within these seemingly homogenous cell populations. Furthermore, we demonstrate that changes in cellular density and levitation profiles can be monitored in real time at single-cell resolution, allowing quantification of heterogeneous temporal responses of each cell to environmental stressors. These data establish density as a powerful biomarker for investigating living systems and their responses. Thereby, our method enables rapid, density-based imaging and profiling of single cells with intriguing applications, such as label-free identification and monitoring of heterogeneous biological changes under various physiological conditions, including antibiotic or cancer treatment in personalized medicine.

Multitarget, quantitative nanoplasmonic electrical field-enhanced resonating device (NE2RD) for diagnostics.

Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients' homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE(2)RD), which addresses all these impediments on a single platform. The NE(2)RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE(2)RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE(2)RD's broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients' homes.

Paper and flexible substrates as materials for biosensing platforms to detect multiple biotargets.

The need for sensitive, robust, portable, and inexpensive biosensing platforms is of significant interest in clinical applications for disease diagnosis and treatment monitoring at the point-of-care (POC) settings. Rapid, accurate POC diagnostic assays play a crucial role in developing countries, where there are limited laboratory infrastructure, trained personnel, and financial support. However, current diagnostic assays commonly require long assay time, sophisticated infrastructure and expensive reagents that are not compatible with resource-constrained settings. Although paper and flexible material-based platform technologies provide alternative approaches to develop POC diagnostic assays for broad applications in medicine, they have technical challenges integrating to different detection modalities. Here, we address the limited capability of current paper and flexible material-based platforms by integrating cellulose paper and flexible polyester films as diagnostic biosensing materials with various detection modalities through the development and validation of new widely applicable electrical and optical sensing mechanisms using antibodies and peptides. By incorporating these different detection modalities, we present selective and accurate capture and detection of multiple biotargets including viruses (Human Immunodeficiency Virus-1), bacteria (Escherichia coli and Staphylococcus aureus), and cells (CD4(+) T lymphocytes) from fingerprick volume equivalent of multiple biological specimens such as whole blood, plasma, and peritoneal dialysis effluent with clinically relevant detection and sensitivity.

Portable microfluidic integrated plasmonic platform for pathogen detection.

Timely detection of infectious agents is critical in early diagnosis and treatment of infectious diseases. Conventional pathogen detection methods, such as enzyme linked immunosorbent assay (ELISA), culturing or polymerase chain reaction (PCR) require long assay times, and complex and expensive instruments, which are not adaptable to point-of-care (POC) needs at resource-constrained as well as primary care settings. Therefore, there is an unmet need to develop simple, rapid, and accurate methods for detection of pathogens at the POC. Here, we present a portable, multiplex, inexpensive microfluidic-integrated surface plasmon resonance (SPR) platform that detects and quantifies bacteria, i.e., Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) rapidly. The platform presented reliable capture and detection of E. coli at concentrations ranging from ~10(5) to 3.2 × 10(7) CFUs/mL in phosphate buffered saline (PBS) and peritoneal dialysis (PD) fluid. The multiplexing and specificity capability of the platform was also tested with S. aureus samples. The presented platform technology could potentially be applicable to capture and detect other pathogens at the POC and primary care settings.

Enhanced efficacy of superparamagnetic iron oxide nanoparticles against antibiotic-resistant biofilms in the presence of metabolites.

Antibiotic resistance and the lack of new antibacterial agents cause major challenges for the treatment of infections. Here, we describe a simple, broad-spectrum, and low-cost dual-sided approach which uses superparamagnetic iron oxide particles (SPION) in combination with fructose metabolites as an alternative to existing antibacterial strategies. This strategy offers further improved efficacy of SPION against persistent gram-positive and gram-negative bacteria infections by manipulating the biofilm metabolic microenvironment and outperforms vancomycin (the antibiotic of last resort), creating a new nanotechnology-driven approach.

Short communication: Carboxylate functionalized superparamagnetic iron oxide nanoparticles (SPION) for the reduction of S. aureus growth post biofilm formation.

Biofilms formed by antibiotic resistant Staphylococcus aureus (S. aureus) continue to be a problem for medical devices. Antibiotic resistant bacteria (such as S. aureus) often complicate the treatment and healing of the patient, yet, medical devices are needed to heal such patients. Therefore, methods to treat these Biofilms once formed on medical devices are badly needed. Due to their small size and magnetic properties, superparamagnetic iron oxide nanoparticles (SPION) may be one possible material to penetrate Biofilms and kill or slow the growth of bacteria. In this study, SPION were functionalized with amine, carboxylate, and isocyanate functional groups to further improve their efficacy to disrupt the growth of S. aureus Biofilms. Without the use of antibiotics, results showed that SPION functionalized with carboxylate groups (followed by isocyanate then amine functional groups then unfunctionalized SPION) significantly disrupted Biofilms and retarded the growth of S. aureus compared to untreated Biofilms (by over 35% after 24 hours).

Eradicating antibiotic-resistant biofilms with silver-conjugated superparamagnetic iron oxide nanoparticles.

Concerns about antibiotic-resistant microorganisms, such as methicillin-resistant Staphylococcus aureus (MRSA), is causing a resurgence in the search for novel strategies which can eradicate infections without the use of antibiotics. In this study, the unique magnetic and antibacterial properties of superparamagnetic iron oxide nanoparticles (SPION) and silver have been combined through the design of silver-conjugated SPION. For the first time, it is demonstrated that MRSA biofilms can be eradicated by silver-conjugated SPION without resorting to the use of antibiotics. A significant decrease in biofilm mass, which corresponds to a seven orders of magnitude decrease in viability, is observed when MRSA biofilms are treated with 1 mg/mL of silver-conjugated SPION (p < 0.01). Moreover, SPION anti-biofilm efficacy is further improved in the presence of an external magnetic field. The anti-biofilm property of silver-conjugated SPION treatment is due to the significant increases in intracellular or membrane-bound iron (p < 0.001), sulfur (p < 0.05), and silver (p < 0.001) concentrations, thus increases in SPION uptake within the biofilms. For this reason, this study demonstrates for the first time that silver-conjugated SPION could be used as a targeted antibacterial therapy to the infection site. Thus, this novel infection eradication strategy holds great promise to be an alternative to the antibiotic of last resort, vancomycin, which bacteria have already started to develop a resistance towards.

Superparamagnetic iron oxide nanoparticles (SPION) for the treatment of antibiotic-resistant biofilms.

Bacterial infections caused by antibiotic-resistant strains are of deep concern due to an increasing prevalence, and are a major cause of morbidity in the United States of America. In particular, medical device failures, and thus human lives, are greatly impacted by infections, where the treatments required are further complicated by the tendency of pathogenic bacteria, such as Staphylococcus aureus, to produce antibiotic resistant biofilms. In this study, a panel of relevant antibiotics used clinically including penicillin, oxacillin, gentamicin, streptomycin, and vancomycin are tested, and although antibiotics are effective against free-floating planktonic S. aureus, either no change in biofilm function is observed, or, more frequently, biofilm function is enhanced. As an alternative, superparamagnetic iron oxide nanoparticles (SPION) are synthesized through a two-step process with dimercaptosuccinic acid as a chelator, followed by the conjugation of metals including iron, zinc, and silver; thus, the antibacterial properties of the metals are coupled to the superparamagnetic properties of SPION. SPION might be the ideal antibacterial treatment, with a superior ability to decrease multiple bacterial functions, target infections in a magnetic field, and had activity better than antibiotics or metal salts alone, as is required for the treatment of medical device infections for which no treatment exists today.