RESUMEN
Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvß6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer's safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin αvß6.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Fibrosis Pulmonar Idiopática/diagnóstico , Integrinas/metabolismo , Neoplasias/diagnóstico , Cristalografía por Rayos X , Voluntarios Sanos , Humanos , Imagen por Resonancia Magnética , Tomografía de Emisión de PositronesRESUMEN
Each immunoglobulin isotype has unique immune effector functions. The contribution of these functions in the elimination of pathogens and tumors can be determined by monitoring quantitative temporal changes in isotype levels. Here, we developed a novel technique using magneto-nanosensors based on the effect of giant magnetoresistance (GMR) for longitudinal monitoring of total and antigen-specific isotype levels with high precision, using as little as 1 nL of serum. Combining in vitro serologic measurements with in vivo imaging techniques, we investigated the role of the antibody response in the regression of firefly luciferase (FL)-labeled lymphoma cells in spleen, kidney, and lymph nodes in a syngeneic Burkitt's lymphoma mouse model. Regression status was determined by whole body bioluminescent imaging (BLI). The magneto-nanosensors revealed that anti-FL IgG2a and total IgG2a were elevated and sustained in regression mice compared to non-regression mice (p < 0.05). This platform shows promise for monitoring immunotherapy, vaccination, and autoimmunity.
Asunto(s)
Formación de Anticuerpos , Técnicas Biosensibles/instrumentación , Linfoma de Burkitt/inmunología , Inmunoglobulina G/análisis , Magnetismo/instrumentación , Animales , Linfoma de Burkitt/sangre , Linfoma de Burkitt/diagnóstico por imagen , Diseño de Equipo , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Mediciones Luminiscentes/métodos , Ratones , Ratones Endogámicos C57BL , Imagen Óptica/instrumentación , Tamaño de la MuestraRESUMEN
Substantial efforts have been made to understand the interactions between immune checkpoint receptors and their ligands targeted in immunotherapies against cancer. To carefully characterize the complete network of interactions involved and the binding affinities between their extracellular domains, an improved kinetic assay is needed to overcome limitations with surface plasmon resonance (SPR). Here, we present a magneto-nanosensor platform integrated with a microfluidic chip that allows measurement of dissociation constants in the micromolar-range. High-density conjugation of magnetic nanoparticles with prey proteins allows multivalent receptor interactions with sensor-immobilized bait proteins, more closely mimicking natural-receptor clustering on cells. The platform has advantages over traditional SPR in terms of insensitivity of signal responses to pH and salinity, less consumption of proteins and better sensitivities. Using this platform, we characterized the binding affinities of the PD-1-PD-L1/PD-L2 co-inhibitory receptor system, and discovered an unexpected interaction between the two known PD-1 ligands, PD-L1 and PD-L2.
Asunto(s)
Antígeno B7-H1/metabolismo , Magnetismo/métodos , Nanopartículas/química , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Mapeo de Interacción de Proteínas , Sistemas de Computación , Humanos , Concentración de Iones de Hidrógeno , Cinética , Unión Proteica , SalinidadRESUMEN
We demonstrate microfluidic partitioning of a giant magnetoresistive sensor array into individually addressable compartments that enhances its effective use. Using different samples and reagents in each compartment enables measuring of cross-reactive species and wide dynamic ranges on a single chip. This compartmentalization technique motivates the employment of high density sensor arrays for highly parallelized measurements in lab-on-a-chip devices.
Asunto(s)
Campos Magnéticos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Técnicas Biosensibles/instrumentación , Inmunoensayo/instrumentación , Dispositivos Laboratorio en un Chip , Proteínas/análisisRESUMEN
We report a case of a 72-year-old man who presented with a persistent pleural effusion and painful abscess in the right lower chest wall 6â months following a laparoscopic cholecystectomy. The patient subsequently developed a chronic cutaneous chest wall fistula requiring a large resection and complex closure. The complication was likely secondary to intraoperative spillage of gallstones. While previous reports describe gallstone spillage in the abdominal cavity as benign, this case illustrates that stones left in the abdominal cavity can potentially lead to significant morbidity. Therefore, stones should be diligently removed from the abdominal cavity when spillage occurs. In addition, it is important that operative notes reflect the occurrence of stone spillage so stones may be suspected when a patient presents with an abdominal or thoracic infection following a cholecystectomy.
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Colecistitis/etiología , Fístula Cutánea/etiología , Cálculos Biliares/complicaciones , Pared Torácica , Anciano , Colecistectomía Laparoscópica/métodos , Colecistitis/diagnóstico , Colecistitis/cirugía , Enfermedad Crónica , Fístula Cutánea/diagnóstico , Fístula Cutánea/cirugía , Diagnóstico Diferencial , Fístula/diagnóstico , Fístula/etiología , Fístula/cirugía , Cálculos Biliares/diagnóstico , Cálculos Biliares/cirugía , Humanos , Masculino , Procedimientos de Cirugía Plástica/métodos , Colgajos Quirúrgicos , Tomografía Computarizada por Rayos XRESUMEN
Detection and characterization of circulating tumor cells (CTCs) may reveal insights into the diagnosis and treatment of malignant disease. Technologies for isolating CTCs developed thus far suffer from one or more limitations, such as low throughput, inability to release captured cells, and reliance on expensive instrumentation for enrichment or subsequent characterization. We report a continuing development of a magnetic separation device, the magnetic sifter, which is a miniature microfluidic chip with a dense array of magnetic pores. It offers high efficiency capture of tumor cells, labeled with magnetic nanoparticles, from whole blood with high throughput and efficient release of captured cells. For subsequent characterization of CTCs, an assay, using a protein chip with giant magnetoresistive nanosensors, has been implemented for mutational analysis of CTCs enriched with the magnetic sifter. The use of these magnetic technologies, which are separate devices, may lead the way to routine preparation and characterization of "liquid biopsies" from cancer patients.
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Separación Celular/métodos , Magnetismo , Técnicas Analíticas Microfluídicas/métodos , Células Neoplásicas Circulantes/metabolismo , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Separación Celular/instrumentación , Molécula de Adhesión Celular Epitelial , Receptores ErbB/genética , Receptores ErbB/inmunología , Receptores ErbB/metabolismo , Fluoresceína-5-Isotiocianato/química , Humanos , Queratinas/inmunología , Queratinas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Células MCF-7 , Nanopartículas de Magnetita/química , Técnicas Analíticas Microfluídicas/instrumentación , MutaciónRESUMEN
Giant magnetoresistive (GMR) nanosensors provide a novel approach for measuring protein concentrations in blood for medical diagnosis. Using an in vivo mouse radiation model, we developed protocols for measuring Flt3 ligand (Flt3lg) and serum amyloid A1 (Saa1) in small amounts of blood collected during the first week after X-ray exposures of sham, 0.1, 1, 2, 3, or 6â Gy. Flt3lg concentrations showed excellent dose discrimination at ≥ 1â Gy in the time window of 1 to 7 days after exposure except 1â Gy at day 7. Saa1 dose response was limited to the first two days after exposure. A multiplex assay with both proteins showed improved dose classification accuracy. Our magneto-nanosensor assay demonstrates the dose and time responses, low-dose sensitivity, small volume requirements, and rapid speed that have important advantages in radiation triage biodosimetry.
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Técnicas Biosensibles , Proteínas Sanguíneas , Nanotecnología , Radiación Ionizante , Radiometría , Animales , Biomarcadores/sangre , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/normas , Relación Dosis-Respuesta en la Radiación , Femenino , Masculino , Proteínas de la Membrana/sangre , Ratones , Reproducibilidad de los Resultados , Proteína Amiloide A Sérica , Factores de TiempoRESUMEN
Surgical competency requires the development of decision-making and technical skills. Despite lectures, literature, and written and oral examinations, both skill sets are difficult to systematically teach and analyze. With the advent of head-mounted video cameras, we seek to incorporate a surgical video database into our surgical training curriculum. We hope to not only change the way and rate at which surgical trainees develop their surgical skills but to also introduce a novel tool for surgical skill assessment.
Asunto(s)
Competencia Clínica , Internado y Residencia , Cirugía Plástica/educación , Grabación en Video/instrumentación , Diseño de Equipo , Internado y Residencia/métodos , Grabación en Video/economíaRESUMEN
BACKGROUND: This study seeks to determine human host response to fetal bovine acellular dermal matrix (ADM) in staged implant-based breast reconstruction. METHODS: A prospective study was performed for patients undergoing immediate breast reconstruction with tissue expander placement and SurgiMend acellular fetal bovine dermis. At the time of exchange for permanent implant, we obtained tissue specimens of SurgiMend and native capsule. Histological and immunohistochemical assays were performed to characterize the extent of ADM incorporation/degradation, host cell infiltration, neovascularization, inflammation, and host replacement of acellular fetal bovine collagen. RESULTS: Seventeen capsules from 12 patients were included in our study. The average "implantation" time of SurgiMend was 7.8 months (range, 2-23 months). Histological analysis of the biopsy of tissue revealed rare infiltration of host inflammatory cells, even at 23 months. One patient had an infection requiring removal of the tissue expander at 2 months. Contracture, inflammatory changes, edema, and polymorphonuclear leukocyte infiltration were rare in the ADM. An acellular capsule was seen in many cases, at the interface of SurgiMend with the tissue expander. CONCLUSIONS: SurgiMend demonstrated a very infrequent inflammatory response. An antibody specific to bovine collagen allowed for direct identification of bovine collagen separate from human collagen. Cellular infiltration and neovascularization of SurgiMend correlated with the quality of the mastectomy skin flap rather than the duration of implantation. Future studies are needed to further characterize the molecular mechanisms underlying tissue incorporation of this product.
Asunto(s)
Dermis Acelular , Implantación de Mama , Implantes de Mama , Dispositivos de Expansión Tisular , Adulto , Animales , Implantación de Mama/métodos , Bovinos , Femenino , Feto , Humanos , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
Magnetic nanotechnologies have shown significant potential in several areas of nanomedicine such as imaging, therapeutics, and early disease detection. Giant magnetoresistive spin-valve (GMR SV) sensors coupled with magnetic nanotags (MNTs) possess great promise as ultra-sensitive biosensors for diagnostics. We report an integrated sensor interface for an array of 256 GMR SV biosensors designed in 0.18 µm CMOS. Arranged like an imager, each of the 16 column level readout channels contains an analog front- end and a compact ΣΔ modulator (0.054 mm2) with 84 dB of dynamic range and an input referred noise of 49 nT/âHz. Performance is demonstrated through detection of an ovarian cancer biomarker, secretory leukocyte peptidase inhibitor (SLPI), spiked at concentrations as low as 10 fM. This system is designed as a replacement for optical protein microarrays while also providing real-time kinetics monitoring.
RESUMEN
Despite significant advances in reconstructive surgery, the repair of massive lumbosacral defects poses significant challenges. When the extent of soft tissue loss, tumor resection, and/or radiation therapy preclude the use of traditional local options, such as gluteal advancement flaps or pedicled thigh flaps, then distant flaps are required. We report a case of a 64-year-old male who presented with a large sacral Marjolin's ulcer secondary to recurrent pilonidal cysts and ulcerations. The patient underwent wide local composite resection, which resulted in a wound measuring 450 cm(2) with exposed rectum and sacrum. The massive defect was successfully covered with a free transverse rectus abdominis myocutaneous flap, providing a well-vascularized skin paddle and obviating the need for a latissimus flap with skin graft. The free-TRAM flap proved to be a very robust flap in this situation and would be one of our flaps of choice for similar defects.
Asunto(s)
Carcinoma de Células Escamosas/cirugía , Colgajos Tisulares Libres , Seno Pilonidal/complicaciones , Procedimientos de Cirugía Plástica/métodos , Recto del Abdomen/trasplante , Neoplasias Cutáneas/cirugía , Nalgas/irrigación sanguínea , Carcinoma de Células Escamosas/etiología , Humanos , Región Lumbosacra , Masculino , Persona de Mediana Edad , Neoplasias Cutáneas/etiologíaRESUMEN
Monitoring the kinetics of protein interactions on a high-density sensor array is vital to drug development and proteomic analysis. Label-free kinetic assays based on surface plasmon resonance are the current gold standard, but they have poor detection limits, suffer from non-specific binding, and are not amenable to high-throughput analyses. Here, we show that magnetically responsive nanosensors that have been scaled to over 100,000 sensors per cm² can be used to measure the binding kinetics of various proteins with high spatial and temporal resolution. We present an analytical model that describes the binding of magnetically labelled antibodies to proteins that are immobilized on the sensor surface. This model is able to quantify the kinetics of antibody-antigen binding at sensitivities as low as 20 zeptomoles of solute.
Asunto(s)
Técnicas Biosensibles/instrumentación , Nanopartículas de Magnetita/química , Análisis por Matrices de Proteínas/instrumentación , Reacciones Antígeno-Anticuerpo , Antígenos de Neoplasias/análisis , Antígeno Carcinoembrionario/análisis , Moléculas de Adhesión Celular/análisis , Molécula de Adhesión Celular Epitelial , Cinética , Modelos Moleculares , Unión Proteica , Proteínas/química , Sensibilidad y Especificidad , Resonancia por Plasmón de Superficie/métodos , Factor A de Crecimiento Endotelial Vascular/análisisRESUMEN
Driven by scientific progress and economic stimulus, medical diagnostics will move to a stage in which straightforward medical diagnoses are independent of physician visits and large centralized laboratories. The future of basic diagnostic medicine will lie in the hands of private individuals. We have taken significant strides towards achieving this goal by developing an autoassembly assay for disease biomarker detection which obviates the need for washing steps and is run on a handheld sensing platform. By coupling magnetic nanotechnology with an array of magnetically responsive nanosensors, we demonstrate a rapid, multiplex immunoassay that eliminates the need for trained technicians to run molecular diagnostic tests. Furthermore, the platform is battery-powered and ultraportable, allowing the assay to be run anywhere in the world by any individual.
Asunto(s)
Técnicas de Laboratorio Clínico/instrumentación , Salud , Internacionalidad , Nanotecnología/instrumentación , Técnicas Biosensibles , Países en Desarrollo , Proteína p24 del Núcleo del VIH/análisis , Inmunoensayo , Magnetismo , Virus/aislamiento & purificaciónRESUMEN
We report an autoassembly protein array capable of rapidly screening for aberrant antibody-antigen binding events. Our technique combines magnetic nanoparticle technology with proximity-based, magnetically responsive nanosensors for rapid (under 15 min) and high-density screening of antibody cross-reactivity at sensitivities down to 50 fM in a homogeneous assay. This method will enable the identification of the precise cause of aberrant or cross-reactive binding events in an easy-to-use, rapid, and high-throughput manner.
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Anticuerpos/análisis , Anticuerpos/inmunología , Reacciones Cruzadas/inmunología , Nanotecnología , Análisis por Matrices de Proteínas , Reacciones Antígeno-Anticuerpo , Ensayos Analíticos de Alto Rendimiento , Inmunoensayo , MagnetismoRESUMEN
This paper presents a hand-held, portable biosensor platform for quantitative biomarker measurement. By combining magnetic nanoparticle (MNP) tags with giant magnetoresistive (GMR) spin-valve sensors, the hand-held platform achieves highly sensitive (picomolar) and specific biomarker detection in less than 20 minutes. The rapid analysis and potential low cost make this technology ideal for point-of-care (POC) diagnostics. Furthermore, this platform is able to detect multiple biomarkers simultaneously in a single assay, creating a promising diagnostic tool for a vast number of applications.
RESUMEN
Advances in biosensor technologies for in vitro diagnostics have the potential to transform the practice of medicine. Despite considerable work in the biosensor field, there is still no general sensing platform that can be ubiquitously applied to detect the constellation of biomolecules in diverse clinical samples (for example, serum, urine, cell lysates or saliva) with high sensitivity and large linear dynamic range. A major limitation confounding other technologies is signal distortion that occurs in various matrices due to heterogeneity in ionic strength, pH, temperature and autofluorescence. Here we present a magnetic nanosensor technology that is matrix insensitive yet still capable of rapid, multiplex protein detection with resolution down to attomolar concentrations and extensive linear dynamic range. The matrix insensitivity of our platform to various media demonstrates that our magnetic nanosensor technology can be directly applied to a variety of settings such as molecular biology, clinical diagnostics and biodefense.
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Bioensayo , Técnicas Biosensibles/instrumentación , Proteínas/metabolismo , Animales , Técnicas Biosensibles/métodos , Antígeno Carcinoembrionario/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética/métodos , Ratones , Nanotecnología/instrumentación , Nanotecnología/métodos , Óptica y Fotónica/instrumentación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Temperatura , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/instrumentación , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Optical observations of 100 nm metallic magnetic nanoparticles are used to study their magnetic field induced self assembly. Chains with lengths of tens of microns are observed to form within minutes at nanoparticle concentrations of 10(10) per mL. Chain rotation and magnetophoresis are readily observed, and SEM reveals that long chains are not simple single particle filaments. Similar chains are detected for several 100 nm commercial bio-separation nanoparticles. We demonstrate the staged magnetic condensation of different types of nanoparticles into composite structures and show that magnetic chains bind to immunomagnetically labeled cells, serving as temporary handles which allow novel magnetic cell manipulations.
RESUMEN
Magnetic nanotags (MNTs) are a promising alternative to fluorescent labels in biomolecular detection assays, because minute quantities of MNTs can be detected with inexpensive giant magnetoresistive (GMR) sensors, such as spin valve (SV) sensors. However, translating this promise into easy to use and multilplexed protein assays, which are highly sought after in molecular diagnostics such as cancer diagnosis and treatment monitoring, has been challenging. Here, we demonstrate multiplex protein detection of potential cancer markers at subpicomolar concentration levels and with a dynamic range of more than four decades. With the addition of nanotag amplification, the analytic sensitivity extends into the low fM concentration range. The multianalyte ability, sensitivity, scalability, and ease of use of the MNT-based protein assay technology make it a strong contender for versatile and portable molecular diagnostics in both research and clinical settings.