RESUMEN
Respirable crystalline silica (RCS) produced in mining and construction industries can cause life-threatening diseases such as silicosis, lung cancer, and chronic obstructive pulmonary disease (COPD). These diseases could be more effectively treated and prevented if RCS-related biomarkers were identified and measured at an early stage of disease progression, which makes development of a point of care test (POCT) platform extremely desirable for early diagnosis. In this work, a new, highly sensitive lab on a chip (LOC) immunoassay has been designed, developed, and characterized for tumor necrosis factor α (TNF-α), a protein biomarker that causes lung inflammation due to RCS exposure. The designed LOC device is composed of four reservoirs for sample, enzyme conjugated detection antibody, wash buffer, and chemiluminescence substrate in liquid form, along with three spiral reaction chambers for test, positive control, and negative control. All reservoirs and spiral microchannels were connected in series and designed to perform sequential delivery of immunoassay reagents with minimal user intervention. The developed LOC measured TNF-α concentrations as low as 16 pg/mL in plasma from RCS-exposed rats and also had a limit of detection (LOD) of 0.5 pg/mL in spiked artificial serum. In addition, the analysis time was drastically reduced to about 30 min, as opposed to hours in conventional methods. Successful implementation of a highly sensitive, chemiluminescence-based immunoassay on a preloaded LOC with proper quality control, as reported in this work, can pave the way toward developing a new rapid POCT platform for in-field clinical diagnosis.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/métodos , Dióxido de Silicio/toxicidad , Silicosis/diagnóstico , Factor de Necrosis Tumoral alfa/sangre , Animales , Anticuerpos Inmovilizados/inmunología , Biomarcadores/sangre , Peroxidasa de Rábano Silvestre/química , Límite de Detección , Sustancias Luminiscentes/química , Mediciones Luminiscentes , Masculino , Técnicas Analíticas Microfluídicas/instrumentación , Pruebas en el Punto de Atención , Ratas Endogámicas F344 , Silicosis/sangre , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Over the last few years, lateral flow assay (LFA) devices have grown to be the most common point-of-care test (POCT) platform facilitating disease diagnostics in low-resource environments. However, the lack of consistency and the limited sensitivity of these devices often lead to misdiagnosis and generates the need for an alternate approach. A chemiluminescence based microchannel-based lateral flow assay (MLFA) in a POCT platform can result in a much higher sensitivity but involves multiple additional steps of liquid reagents for the sequential execution of the signal amplification protocol. One of the best ways to develop a sample-to-answer system with minimum user intervention is to dry reagents on a chip prior to sample addition and to control the flow of the biological fluid through the drying chambers resulting in the reconstitution of the reagents. This work reports the methods for the successful lyophilization of the chemiluminescent substrate and its reconstitution in artificial serum without any significant loss of functionality. The lyophilized reagents were reconstituted and incorporated into the reaction chambers of a designed polymer lab-on-a-chip to implement a sandwich assay for the detection of malarial biomarkers. The results report a limit of detection (LOD) of 5.75 ng mL-1 which is sensitive enough to detect active malarial infection. Successful lyophilization and reconstitution of the chemiluminescent substrate, as reported here, can pave the way towards developing an autonomous POCT system implementing chemiluminescence based sandwich ELISA for enhanced sensitivity, portability, and ease-of-use in resource limited settings.
Asunto(s)
Bioensayo/instrumentación , Dispositivos Laboratorio en un Chip/normas , Mediciones Luminiscentes/métodos , Técnicas Analíticas Microfluídicas/métodos , Sistemas de Atención de Punto , Pruebas en el Punto de Atención/normas , Ensayo de Inmunoadsorción Enzimática , Liofilización , Humanos , Límite de Detección , Especificidad por SustratoRESUMEN
A specific double-stranded DNA sensing system is of great interest for diagnostic and other biomedical applications. Zinc finger domains, which recognize double-stranded DNA, can be engineered to form custom DNA-binding proteins for the recognition of specific DNA sequences. As a proof of concept, a sequence-enabled reassembly of a TEM-1 ß-lactamase system (SEER-LAC) was previously demonstrated to develop zinc finger protein (ZFP) arrays for the detection of a double-stranded bacterial DNA sequence. Here, we implemented the SEER-LAC system to demonstrate the direct detection of pathogen-specific DNA sequences present in E. coli O157:H7 on a lab-on-a-chip. ZFPs custom-designed to detect Shiga toxin in E. coli O157:H7 were immobilized on a cyclic olefin copolymer (COC) chip, which can function as a non-PCR based molecular diagnostic device. Pathogen-specific double-stranded DNA was directly detected by using engineered ZFPs immobilized on the COC chip with high specificity, providing a detection limit of 10 fmol of target DNA in a colorimetric assay. Therefore, in this study, we demonstrated the great potential of ZFP arrays on the COC chip for further development of a simple and novel lab-on-a-chip technology for the detection of pathogens.
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ADN Bacteriano/aislamiento & purificación , Proteínas de Unión al ADN/química , Escherichia coli O157/aislamiento & purificación , Proteínas Inmovilizadas/química , Dedos de Zinc , Escherichia coli O157/genética , Dispositivos Laboratorio en un Chip , Polímeros , Ingeniería de Proteínas , Sensibilidad y EspecificidadRESUMEN
Real-time imaging of fluorescent reporters plays a critical role in elucidating fundamental molecular mechanisms including circadian rhythms in the model filamentous fungus, Neurospora crassa. However, monitoring N. crassa for an extended period of time with single nucleus resolution is a technically challenging task due to hyphal growth that rapidly moves beyond a region of interest during microscopy experiments. In this report, we have proposed a two-dimensional spiral-based microfluidic platform and applied for monitoring the single-nucleus dynamics in N. crassa for long-term time course experiments.
Asunto(s)
Dispositivos Laboratorio en un Chip , Neurospora crassa/ultraestructura , Núcleo Celular/ultraestructura , Ritmo CircadianoRESUMEN
We present a microfabricated neural catheter for real-time continuous monitoring of multiple physiological, biochemical and electrophysiological variables that are critical to the diagnosis and treatment of evolving brain injury. The first generation neural catheter was realized by polyimide-based micromachining and a spiral rolling packaging method. The mechanical design and electrical operation of the microsensors were optimized and tailored for multimodal monitoring in rat brain such that the potential thermal, chemical and electrical crosstalk among the microsensors as well as errors from micro-environmental fluctuations are minimized. In vitro cytotoxicity analyses suggest that the developed neural catheters are minimally toxic to rat cortical neuronal cultures. In addition, in vivo histopathology results showed neither acute nor chronic inflammation for 7 days post implantation. The performance of the neural catheter was assessed in an in vivo needle prick model as a translational replica of a "mini" traumatic brain injury. It successfully monitored the expected transient brain oxygen, temperature, regional cerebral blood flow, and DC potential changes during the passage of spreading depolarization waves. We envisage that the developed multimodal neural catheter can be used to decipher the causes and consequences of secondary brain injury processes with high spatial and temporal resolution while reducing the potential for iatrogenic injury inherent to current use of multiple invasive probes.
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Lesiones Encefálicas/fisiopatología , Catéteres , Depresión de Propagación Cortical , Electrodos Implantados , Resinas Sintéticas , Animales , Lesiones Encefálicas/patología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Cerebral blood flow (CBF) plays a critical role in the exchange of nutrients and metabolites at the capillary level and is tightly regulated to meet the metabolic demands of the brain. After major brain injuries, CBF normally decreases and supporting the injured brain with adequate CBF is a mainstay of therapy after traumatic brain injury. Quantitative and localized measurement of CBF is therefore critically important for evaluation of treatment efficacy and also for understanding of cerebral pathophysiology. We present here an improved thermal flow microsensor and its operation which provides higher accuracy compared to existing devices. The flow microsensor consists of three components, two stacked-up thin film resistive elements serving as composite heater/temperature sensor and one remote resistive element for environmental temperature compensation. It operates in constant-temperature mode (~2 °C above the medium temperature) providing 20 ms temporal resolution. Compared to previous thermal flow microsensor based on self-heating and self-sensing design, the sensor presented provides at least two-fold improvement in accuracy in the range from 0 to 200 ml/100 g/min. This is mainly achieved by using the stacked-up structure, where the heating and sensing are separated to improve the temperature measurement accuracy by minimization of errors introduced by self-heating.
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Velocidad del Flujo Sanguíneo/fisiología , Circulación Cerebrovascular/fisiología , Conductometría/instrumentación , Reología/instrumentación , Termografía/instrumentación , Transductores , Animales , Diseño Asistido por Computadora , Diseño de Equipo , Análisis de Falla de Equipo , Calefacción/instrumentación , Masculino , Miniaturización , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
A new micro blood sampling system has been designed, fabricated, and characterized to reduce iatrogenic blood loss from the catheterized neonates and pediatrics in intensive care unit by providing micro-volume of blood to analytical biomedical microdevices which can do point-of-care testing for their critical care. The system can not only save enormous iatrogenic blood loss through 1 to 10 µL of blood sampling and re-infusion of 1 to 5 mL of discard blood but also reduce the infection risk through the closed structure while satisfying the key criteria of the blood sampler. The sampled blood preserved its quality without rupturing of red blood cells verified by blood potassium concentrations of 3.86 ± 0.07 mM on the sampled blood which is similar to 3.81 ± 0.04 mM measured from the blood which did not go through the system. The sampling volume among the sampling channels showed consistency with the relative standard deviation of 1.41 %. In addition to the micro blood sampling capability, the sampling system showed negligible sample cross-contamination. The analyte-free samples collected after aspirating 7,500 times higher signal sample showed the same output signal as blank. The system was also demonstrated not to cause air-embolism by having no bubble generation during flushing procedure and the system was verified as leak-free since there was no fluid leakage under 30 times higher pressure than central venous pressure for 24 h.
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Recolección de Muestras de Sangre/instrumentación , Catéteres Venosos Centrales , Unidades de Cuidado Intensivo Neonatal , Unidades de Cuidado Intensivo Pediátrico , Microfluídica/instrumentación , Manejo de Especímenes/instrumentación , Niño , Preescolar , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Lactante , Recién Nacido , MasculinoRESUMEN
Retraction of 'A new polymer lab-on-a-chip (LOC) based on a microfluidic capillary flow assay (MCFA) for detecting unbound cortisol in saliva' by Vinitha T. U. et al., Lab Chip, 2020, 20, 1961-1974, DOI: https://doi.org/10.1039/D0LC00071J.
RESUMEN
An efficient method to form lipid bilayers inside an array of microfluidic channels has been developed and applied to monitor the membrane-embedded phi29 DNA packaging motor with an electrochemical characterization on a lab-on-a-chip (LOC) platform. A push-pull junction capturing approach was applied to confine a small amount of the lipid solution inside a microchannel. The selective permeability between solvents and water in PDMS was utilized to extract the solvent from the lipid solution, resulting in a self-formation of the lipid bilayer in the microchannel array. Each microchannel was independently connected to a silver/silver chloride (Ag/AgCl) electrode array, leading to a high-throughput monitoring of the nanopore insertion in the formed lipid bilayers. The formation of multiple lipid bilayers inside an array of microchannels and the simultaneous electrical and optical monitoring of multiple bilayer provides an efficient LOC platform for the further development of single phi29 motor pore sensing and high throughput single pore dsDNA sequencing.
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Empaquetamiento del ADN , Membrana Dobles de Lípidos/química , Microfluídica/instrumentación , Microfluídica/métodos , Nanoporos/ultraestructura , Fagos de Bacillus/química , Técnicas Biosensibles , Electrodos , Diseño de Equipo , Secuenciación de Nucleótidos de Alto Rendimiento , Técnicas Analíticas Microfluídicas/métodos , Análisis de Secuencia de ADN , Compuestos de Plata/metabolismo , Solventes/química , Proteínas Virales/química , Agua/químicaRESUMEN
The injured brain is vulnerable to increases in temperature after severe head injury. Therefore, accurate and reliable measurement of brain temperature is important to optimize patient outcome. In this work, we have fabricated, optimized and characterized temperature sensors for use with a micromachined smart catheter for multimodal intracranial monitoring. Developed temperature sensors have resistance of 100.79 ± 1.19Ω and sensitivity of 67.95 mV/°C in the operating range from15-50°C, and time constant of 180 ms. Under the optimized excitation current of 500 µA, adequate signal-to-noise ratio was achieved without causing self-heating, and changes in immersion depth did not introduce clinically significant errors of measurements (<0.01°C). We evaluated the accuracy and long-term drift (5 days) of twenty temperature sensors in comparison to two types of commercial temperature probes (USB Reference Thermometer, NIST-traceable bulk probe with 0.05°C accuracy; and IT-21, type T type clinical microprobe with guaranteed 0.1°C accuracy) under controlled laboratory conditions. These in vitro experimental data showed that the temperature measurement performance of our sensors was accurate and reliable over the course of 5 days. The smart catheter temperature sensors provided accuracy and long-term stability comparable to those of commercial tissue-implantable microprobes, and therefore provide a means for temperature measurement in a microfabricated, multimodal cerebral monitoring device.
Asunto(s)
Temperatura Corporal , Encéfalo/fisiología , Termómetros , Catéteres , Humanos , Relación Señal-RuidoRESUMEN
This work describes the development of a micromachined lab-on-a-tube device for simultaneous measurement of brain temperature and regional cerebral blood flow. The device consists of two micromachined gold resistance temperature detectors with a 4-wire configuration. One is used as a temperature sensor and the other as a flow sensor. The temperature sensor operates with AC excitation current of 500 µA and updates its outputs at a rate of 5 Hz. The flow sensor employs a periodic heating and cooling technique under constant-temperature mode and updates its outputs at a rate of 0.1 Hz. The temperature sensor is also used to compensate for temperature changes during the heating period of the flow sensor to improve the accuracy of flow measurements. To prevent thermal and electronic crosstalk between the sensors, the temperature sensor is located outside the "thermal influence" region of the flow sensor and the sensors are separated into two different layers with a thin-film Copper shield. We evaluated the sensors for accuracy, crosstalk and long-term drift in human blood-stained cerebrospinal fluid. These in vitro experiments showed that simultaneous temperature and flow measurements with a single lab-on-a-tube device are accurate and reliable over the course of 5 days. It has a resolution of 0.013 °C and 0.18 ml/100 g/min; and achieves an accuracy of 0.1 °C and 5 ml/100 g/min for temperature and flow sensors respectively. The prototype device and techniques developed here establish a foundation for a multi-sensor lab-on-a-tube, enabling versatile multimodality monitoring applications.
Asunto(s)
Encéfalo/irrigación sanguínea , Circulación Cerebrovascular , Microtecnología/instrumentación , Temperatura , Factores de TiempoRESUMEN
This work seeks to extend the utility of microfluidics to conventional blood sampling aperati. Daily medical care of hospitalized patients demands repeated needle punctures or interfacing with a catheter to collect blood samples. Large, research grade systems can autonomously sample blood from laboratory animals; however, a disposable aperatus that can be used to repeatedly sample blood from hospitalized patients does not exist. We have designed, fabricated and demonstrated a 3-layered rigid polymer microfluidic blood sampling device with integrated polymer pinch valves for placement in-line between a patient and a saline infusion system. The blood sampler we designed seeks to mitigate sample cross contamination, reduce risks of microbial contamination associated with invasive blood sampling and improve technical ease of blood sampling. Clinical laboratory tests and microfluidic devices for rapid point-of-care-testing (POCT) of patient samples require human sampling procedures for collection of a patient sample at defined time points. The microfluidic sampling device is designed ultimately to be backwards compatible with existing clinical saline infusion protocols and function as a universal front-end blood sampling unit for the variety of microfluidic lab chips and POCT devices.
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Recolección de Muestras de Sangre/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Microfluídica/instrumentación , Recolección de Muestras de Sangre/métodos , Catéteres , Diseño de Equipo , Humanos , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Microtecnología , Sistemas de Atención de Punto , Punciones/instrumentación , Punciones/métodos , Cloruro de SodioRESUMEN
This technical note presents a simple and disposable lab-on-a-tube (LOT) for point-of-care measurements of multiple analytes. LOT is a one-step device that can perform both sample collection and multi-sensing on-site. Sample collection is conducted by taking advantage of its inherent micro/macro channel structure while multi-sensing is conducted by integrated microsensors. This approach ensures reliable transportation of various samples into the testing area by either passive capillary force or active suction force, thus avoiding the need for a pump or injection components as used in lab-on-a-chip systems. The developed LOT (Diameter = 1 mm, Sensing length = 4.5 mm, Required sample volume = 3.5 microl) is capable of simultaneously quantifying the concentrations of glucose, lactate and oxygen in human serum samples. The result suggests the LOT hold great potential for many point-of-care applications.
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Técnicas Biosensibles/instrumentación , Mezclas Complejas/química , Mezclas Complejas/aislamiento & purificación , Técnicas Analíticas Microfluídicas/instrumentación , Sistemas de Atención de Punto , Manejo de Especímenes/instrumentación , Diseño de Equipo , Análisis de Falla de EquipoRESUMEN
A disposable on-chip whole blood/plasma separator, which is able to separate plasma from whole human blood by capillary force through a bead-packed microchannel, has been designed, fabricated and characterized in this work. Various sizes of silica beads were slurry-packed through a microchannel using a bump structure which held beads in a defined region. The bead-packed microchannel induces a capillary force which allows plasma to move forward through the bead-packed column more rapidly than red blood cells (RBCs). The blood/plasma separator with bead-packed microchannel has successfully separated plasma from the whole blood without haemolysis of RBCs. The separation method developed in this work can be applied to various on-chip stationary filtrations of RBC for point-of-care clinical diagnostics.
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Recolección de Muestras de Sangre/instrumentación , Recolección de Muestras de Sangre/métodos , Cicloparafinas/química , Procedimientos Analíticos en Microchip/métodos , Microesferas , Plasma , Polímeros/química , Humanos , Movimiento , Tamaño de la Partícula , Sistemas de Atención de Punto , Dióxido de Silicio/químicaRESUMEN
A self-aligned nanogap between multiple metal layers has been developed using a new controlled undercut and metallization technique (CUMT), and practically applied for self-assembly of individual carbon nanotubes (CNTs) over the developed nanogap. This new method allows conventional optical lithography to fabricate nanogap electrodes and self-aligned patterns with nanoscale precision. The self-aligned nickel (Ni) pattern on the nanogap electrode works as an assembly spot where the residual iron (Fe) catalyst at the end of the CNT is magnetically captured. The captured CNT is forced to be aligned parallel to the flow direction by fluidic shear force. The combined forces of magnetic attraction and fluidic alignment provide massive self-assembly of CNTs at target positions. Both multiwalled nanotubes (MWNTs) and single walled nanotubes (SWNTs) were successfully assembled over the nanogap electrodes, and their electrical characteristics were fully characterized. The CNTs self-assembled on the developed electrodes with a nanogap and showed a very reliable and reproducible current-voltage (I-V) characteristic. The method developed in this work can envisage the mass fabrication of individual CNT-assembled devices which can be applied to nanoelectronic devices or nanobiosensors.
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Magnetismo , Nanotecnología/métodos , Nanotubos de Carbono/química , Conductividad Eléctrica , Electrodos , Microscopía Electrónica de RastreoRESUMEN
Unbound cortisol in saliva, which can be detected with non-invasive sampling, is now considered as one of the most effective biomarkers for the biochemical evaluation of common mental disorders. In this work, a new polymer lab-on-a-chip (LOC) based on a microfluidic capillary flow assay (MCFA) with on-chip dried reagents was newly developed and fully characterized for the detection of unbound cortisol in saliva. The new MCFA device consisted of serially connected microchannels for sample loading, dried detection antibodies, time delay for incubation time control, a spiral reaction chamber for testing, positive and negative controls, and a capillary pump for waste fluid collection. In addition, a portable fluorescence analyzer was also developed for the rapid quantitative measurement of salivary cortisol with high accuracy. A linear dynamic range of 7.0 pg mL-1-16.0 ng mL-1 was achieved from spiked artificial saliva samples with an inter-chip CV of around 4.0% using the developed LOC and fluorescence analyzer. The achieved results support the effective biochemical analysis of common mental disorders such as chronic stress, depression, anxiety and post traumatic stress disorder (PTSD). The new LOC based on a microfluidic capillary flow assay (MCFA) developed in this work can be one of the most promising LOC platforms for high-sensitivity and quantitative POCT with saliva and blood plasma/serum samples.
Asunto(s)
Hidrocortisona , Saliva , Humanos , Dispositivos Laboratorio en un Chip , Microfluídica , PolímerosRESUMEN
There has been a considerable development in microfluidic based immunodiagnostics over the past few years which has greatly favored the growth of novel point-of-care-testing (POCT). However, the realization of an inexpensive, low-power POCT needs cheap and disposable microfluidic devices that can perform autonomously with minimum user intervention. This work, for the first time, reports the development of a new microchannel capillary flow assay (MCFA) platform that can perform chemiluminescence based ELISA with lyophilized chemiluminescent reagents. This new MCFA platform exploits the ultra-high sensitivity of chemiluminescent detection while eliminating the shortcomings associated with liquid reagent handling, control of assay sequence and user intervention. The functionally designed microchannels along with adequate hydrophilicity produce a sequential flow of assay reagents and autonomously performs the ultra-high sensitive chemiluminescence based ELISA for the detection of malaria biomarker such as PfHRP2. The MCFA platform with no external flow control and simple chemiluminescence detection can easily communicate with smartphone via USB-OTG port using a custom-designed optical detector. The use of the smartphone for display, data transfer, storage and analysis, as well as the source of power allows the development of a smartphone based POCT analyzer for disease diagnostics. This paper reports a limit of detection (LOD) of 8 ng/mL by the smartphone analyzer which is sensitive enough to detect active malarial infection. The MCFA platform developed with the smartphone analyzer can be easily customized for different biomarkers, so a hand-held POCT for various infectious diseases can be envisaged with full networking capability at low cost.
RESUMEN
In this work, a new method of rapidly fabricating thermopolymer and elastomer microfluidic channels has been developed and characterized for production of microfluidics with fixed aspect ratio and 3D tapered channels. A unique way to attain a desired channel depth by simply altering channel width is demonstrated. This rapid prototyping method is compatible with replication methods such as injection molding, hot embossing and elastomer casting and offers the ability to fabricate multiple channel depths (5 microm-1 mm) simultaneously in a single lithographic step. This method yields facile fabrication of 3-dimensionally tapered channels and polymer lab chips.
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Técnicas Analíticas Microfluídicas/instrumentación , Microfluídica/métodos , Diseño de Equipo , Técnicas Analíticas Microfluídicas/economía , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/economía , Microfluídica/instrumentación , Factores de TiempoRESUMEN
A novel lab-on-a-tube integrated with spirally-rolled pressure, temperature, oxygen and glucose microsensors is described for multimodal neuromonitoring of patients with traumatic brain injury. In addition to measuring various crucial parameters in real-time continuous formats, the newly developed device also works as an intraventricular catheter to lower the elevated intracranial pressure by draining cerebrospinal fluid.
Asunto(s)
Lesiones Encefálicas/diagnóstico , Lesiones Encefálicas/fisiopatología , Dispositivos Laboratorio en un Chip , Procedimientos Analíticos en Microchip/métodos , Técnicas Biosensibles , Lesiones Encefálicas/sangre , Lesiones Encefálicas/líquido cefalorraquídeo , Humanos , Monitoreo Fisiológico/instrumentación , Monitoreo Fisiológico/métodos , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
This paper presents an on-chip magnetic cell sorting system for the sorting of cells based on a variety of surface markers. A polymer lab on a chip integrated with an electroplated array of Ni/Fe permalloy has been designed, fabricated, and characterized for the separation of cell substitutes at a variety of flow rates and incubation times. The system sequentially labels cell substitutes with magnetic beads and sorts them, repeating this process to sort for a variety of surface markers. Flow rates and incubation times were varied to characterize the system and produce the best combination of high specific capture and low nonspecific capture. The separation system developed on polymer is selective and efficient while being low cost, portable, and fabricated in a modular structure that can be integrated with other cell handling processes.