RESUMO
Mosquito-borne pathogens plague much of the world, yet rapid and simple diagnosis is not available for many affected patients. Using a custom stereolithography 3D printer, we created microfluidic devices with affinity monoliths that could retain, noncovalently attach a fluorescent tag, and detect oligonucleotide and viral RNA. We optimized the fluorescent binding and sample load times using an oligonucleotide sequence from chikungunya virus (CHIKV). We also tested the specificity of CHIKV capture relative to genetically similar Sindbis virus. Moreover, viral RNA from both viruses was flowed through capture columns to study the efficiency and specificity of the column for viral CHIKV. We detected ~107 loaded viral genome copies, which was similar to levels in clinical samples during acute infection. These results show considerable promise for development of this platform into a rapid mosquito-borne viral pathogen detection system.
Assuntos
Febre de Chikungunya , Vírus Chikungunya , Animais , Humanos , Febre de Chikungunya/diagnóstico , Microfluídica , Vírus Chikungunya/genética , Vírus Chikungunya/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Oligonucleotídeos , Impressão TridimensionalRESUMO
In an effort to develop biomarker-based diagnostics for preterm birth (PTB) risk, we created 3D printed microfluidic devices with multiplexed immunoaffinity monoliths to selectively extract multiple PTB biomarkers. The equilibrium dissociation constant for each monoclonal antibody toward its target PTB biomarker was determined. We confirmed the covalent attachment of three different individual antibodies to affinity monoliths using fluorescence imaging. Three different PTB biomarkers were successfully extracted from human blood serum using their respective single-antibody columns. Selective binding of each antibody toward its target biomarker was observed. Finally, we extracted and eluted three PTB biomarkers from depleted human blood serum in multiplexed immunoaffinity columns in 3D printed microfluidic devices. This is the first demonstration of multiplexed immunoaffinity extraction of PTB biomarkers in 3D printed microfluidic devices.
Assuntos
Dispositivos Lab-On-A-Chip , Nascimento Prematuro , Biomarcadores , Humanos , Recém-Nascido , Nascimento Prematuro/diagnóstico , Impressão Tridimensional , SoroRESUMO
In this work, we demonstrate for the first time the design and fabrication of microchip electrophoresis devices containing cross-shaped channels and spiral electrodes around the separation channel for microchip electrophoresis and capacitively coupled contactless conductivity detection. The whole device was prepared in a digital light processing-based 3D printer in poly(ethylene glycol) diacrylate resin. Outstanding X-Y resolution of the customized 3D printer ensured the fabrication of 40-µm cross section channels. The spiral channels were filled with melted gallium to form conductive electrodes around the separation channel. We demonstrate the applicability of the device on the separation of sodium, potassium, and lithium cations by microchip electrophoresis. Graphical abstract.
RESUMO
Microbial resistance to currently available antibiotics poses a great threat in the global fight against infections. An important step in determining bacterial antibiotic resistance can be selective DNA sequence capture and fluorescence labeling. In this paper, we demonstrate the fabrication of simple, robust, inexpensive microfluidic devices for DNA capture and fluorescence detection of a model antibiotic resistance gene sequence. We laser micromachined polymethyl methacrylate microchannels and enclosed them using pressure-sensitive adhesive tapes. We then formed porous polymer monoliths with DNA capture probes in these microchannels and used them for sequence-specific capture, fluorescent labeling, and laser-induced fluorescence detection of picomolar (pM) concentrations of synthetic and plasmid antibiotic resistance gene targets. The relative fluorescence for the elution peaks increased with loaded target DNA concentration. We observed higher fluorescence signal and percent recovery for synthetic target DNA compared to plasmid DNA at the same loaded target concentration. A non-target gene was used for control experiments and produced < 3% capture relative to the same concentration of target. The full analysis process including device fabrication was completed in less than 90 min with a limit of detection of 30 pM. The simplicity of device fabrication and good DNA capture selectivity demonstrated herein have potential for application with processes for bacterial plasmid DNA extraction and single-particle counting to facilitate determination of antibiotic susceptibility. Graphical abstract.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Dispositivos Lab-On-A-Chip , Plasmídeos/genética , Sondas de DNA/genética , Desenho de Equipamento , Infecções por Escherichia coli/microbiologia , Fluorescência , Humanos , Hibridização de Ácido Nucleico/métodos , Porosidade , Pressão , Sepse/microbiologiaRESUMO
Solid-phase extraction (SPE) is a general preconcentration method for sample preparation that can be performed on a variety of specimens. The miniaturization of SPE within a 3D printed microfluidic device further allows for fast and simple extraction of analytes while also enabling integration of SPE with other sample preparation and separation methods. Here, we present the development and application of a reversed-phase lauryl methacrylate-based monolith, formed in 3D printed microfluidic devices, which can selectively retain peptides and proteins. The effectiveness of these SPE monoliths and 3D printed microfluidic devices was tested using a panel of nine preterm birth biomarkers of varying hydrophobicities and ranging in mass from 2 to 470 kDa. The biomarkers were selectively retained, fluorescently labeled, and eluted separately from the excess fluorescent label in 3D printed microfluidic systems. These are the first results demonstrating microfluidic analysis processes on a complete panel of preterm birth biomarkers, an important step toward developing a miniaturized, fully integrated analysis system.
Assuntos
Fluorescência , Dispositivos Lab-On-A-Chip , Nascimento Prematuro/diagnóstico , Impressão Tridimensional , Extração em Fase Sólida , Biomarcadores/análise , Corantes Fluorescentes/química , HumanosRESUMO
Preterm birth (PTB) related health problems take over one million lives each year, and currently, no clinical analysis is available to determine if a fetus is at risk for PTB. Here, we describe the preparation of a key PTB risk biomarker, thrombin-antithrombin (TAT), and characterize it using dot blots, MS, and microchip electrophoresis (µCE). The pH for fluorescently labeling TAT was also optimized using spectrofluorometry and spectrophotometry. The LOD of TAT was measured in µCE. Lastly, TAT was combined with six other PTB risk biomarkers and separated in µCE. The ability to make and characterize TAT is an important step toward the development of an integrated microfluidic diagnostic for PTB risk.
Assuntos
Antitrombina III/análise , Eletroforese em Microchip/métodos , Espectrometria de Massas/métodos , Peptídeo Hidrolases/análise , Biomarcadores , Humanos , Limite de Detecção , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
Preterm birth (PTB) is defined as birth before the 37th week of pregnancy and results in 15 million early deliveries worldwide every year. Presently, there is no clinical test to determine PTB risk; however, a panel of nine biomarkers found in maternal blood serum has predictive power for a subsequent PTB. A significant step in creating a clinical diagnostic for PTB is designing an automated method to extract and purify these biomarkers from blood serum. Here, microfluidic devices with 45 µm × 50 µm cross-section channels were 3D printed with a built-in polymerization window to allow a glycidyl methacrylate monolith to be site-specifically polymerized within the channel. This monolith was then used as a solid support to attach antibodies for PTB biomarker extraction. Using these functionalized monoliths, it was possible to selectively extract a PTB biomarker, ferritin, from buffer and a human blood serum matrix. This is the first demonstration of monolith formation in a 3D printed microfluidic device for immunoaffinity extraction. Notably, this work is a crucial first step toward developing a 3D printed microfluidic clinical diagnostic for PTB risk.
Assuntos
Dispositivos Lab-On-A-Chip , Gravidez/sangue , Nascimento Prematuro , Impressão Tridimensional/instrumentação , Biomarcadores/sangue , Feminino , Humanos , Recém-Nascido , PolimerizaçãoRESUMO
Preterm birth (PTB) is responsible for over one million infant deaths annually worldwide. Often, the first and only indication of PTB risk is the onset of early labor. Thus, there is an urgent need for an early PTB risk diagnostic that is inexpensive, reliable, and robust. Here, we describe the development of a microchip electrophoresis (µCE) method for separating a mixture of six PTB protein and peptide biomarkers present in maternal blood serum. µCE devices were photografted with a poly(ethylene glycol) diacrylate surface coating to regulate EOF and reduce nonspecific analyte adsorption. Separation conditions including buffer pH, buffer concentration, and applied electric field were varied to improve biomarker peak resolution while minimizing deleterious effects like Joule heating. In this way, it was possible to separate six PTB biomarkers, the first µCE separation of this biomarker panel. LODs were also measured for each of the six PTB biomarkers. In the future, this µCE separation can be integrated with upstream maternal blood serum sample preparation steps to yield a complete PTB risk diagnosis microdevice.
Assuntos
Biomarcadores/sangue , Proteínas Sanguíneas/análise , Eletroforese em Microchip/métodos , Peptídeos/sangue , Nascimento Prematuro/sangue , Feminino , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Polietilenoglicóis/química , Gravidez , Soro/química , Propriedades de SuperfícieRESUMO
Additive manufacturing technology is an emerging method for rapid prototyping, which enables the creation of complex geometries by one-step fabrication processes through a layer-by-layer approach. The simplified fabrication achieved with this methodology opens the way towards a more efficient industrial production, with applications in a great number of fields such as biomedical devices. In biomedicine, blood is the gold-standard biofluid for clinical analysis. However, blood cells generate analytical interferences in many test procedures; hence, it is important to separate plasma from blood cells before analytical testing of blood samples. In this research, a custom-made resin formulation combined with a high-resolution 3D printing methodology were used to achieve a methodology for the fast prototype optimization of an operative plasma separation modular device. Through an iterative process, 17 different prototypes were designed and fabricated with printing times ranging from 5 to 12 min. The final device was evaluated through colorimetric analysis, validating this fabrication approach for the qualitative assessment of plasma separation from whole blood. The 3D printing method used here demonstrates the great contribution that this microfluidic technology will bring to the plasma separation biomedical devices market.
RESUMO
Emerging antimicrobial drug resistance is increasing the complexity involved in treating critical conditions such as bacterial induced sepsis. Methods for diagnosing specific drug resistance tend to be rapid or sensitive, but not both. Detection methods like sequence-specific single-molecule analysis could address this concern if they could be adapted to work on smaller targets similar to those produced in traditional clinical situations. In this work we demonstrate that a 120 bp double stranded polynucleotide with an overhanging single stranded 25 bp probe sequence can be created by immobilizing DNA with a biotin/streptavidin magnetic bead system, labeling with SYBR Gold, and rinsing the excess away while the probe retains multiple fluorophores. These probes with multiple fluorophores can then be used to label a bacterial plasmid target in a sequence-specific manner. These probes enabled the detection of 1 pM plasmid samples containing a portion of an antibiotic resistance gene sequence. This system shows the possibility of improving capture and fluorescence labeling of small nucleic acid fragments, generating lower limits of detection for clinically relevant samples while maintaining rapid processing times.