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3D printers utilize cutting-edge technologies to create three-dimensional objects and are attractive tools for engineering compact microfluidic platforms with complex architectures for chemical and biochemical analyses. 3D printing's popularity is associated with the freedom of creating intricate designs using inexpensive instrumentation, and these tools can produce miniaturized platforms in minutes, facilitating fabrication scaleup. This work discusses key challenges in producing three-dimensional microfluidic structures using currently available 3D printers, addressing considerations about printer capabilities and software limitations encountered in the design and processing of new architectures. This article further communicates the benefits of using three-dimensional structures, including the ability to scalably produce miniaturized analytical systems and the possibility of combining them with multiple processes, such as mixing, pumping, pre-concentration, and detection. Besides increasing analytical applicability, such three-dimensional architectures are important in the eventual design of commercial devices since they can decrease user interferences and reduce the volume of reagents or samples required, making assays more reliable and rapid. Moreover, this manuscript provides insights into research directions involving 3D-printed microfluidic devices. Finally, this work offers an outlook for future developments to provide and take advantage of 3D microfluidic functionality in 3D printing. Graphical abstract Creating three-dimensional microfluidic structures using 3D printing will enable key advances and novel applications in (bio)chemical analysis.
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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.
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Fiebre Chikungunya , Virus Chikungunya , Animales , Humanos , Fiebre Chikungunya/diagnóstico , Microfluídica , Virus Chikungunya/genética , Virus Chikungunya/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Oligonucleótidos , Impresión TridimensionalRESUMEN
Single-cell proteomics (SCP) has great potential to advance biomedical research and personalized medicine. The sensitivity of such measurements increases with low-flow separations (<100 nL/min) due to improved ionization efficiency, but the time required for sample loading, column washing, and regeneration in these systems can lead to low measurement throughput and inefficient utilization of the mass spectrometer. Herein, we developed a two-column liquid chromatography (LC) system that dramatically increases the throughput of label-free SCP using two parallel subsystems to multiplex sample loading, online desalting, analysis, and column regeneration. The integration of MS1-based feature matching increased proteome coverage when short LC gradients were used. The high-throughput LC system was reproducible between the columns, with a 4% difference in median peptide abundance and a median CV of 18% across 100 replicate analyses of a single-cell-sized peptide standard. An average of 621, 774, 952, and 1622 protein groups were identified with total analysis times of 7, 10, 15, and 30 min, corresponding to a measurement throughput of 206, 144, 96, and 48 samples per day, respectively. When applied to single HeLa cells, we identified nearly 1000 protein groups per cell using 30 min cycles and 660 protein groups per cell for 15 min cycles. We explored the possibility of measuring cancer therapeutic targets with a pilot study comparing the K562 and Jurkat leukemia cell lines. This work demonstrates the feasibility of high-throughput label-free single-cell proteomics.
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Péptidos , Proteoma , Cromatografía Liquida/métodos , Células HeLa , Humanos , Péptidos/análisis , Proyectos Piloto , Proteoma/análisisRESUMEN
Liquid microdroplet resonators provide an excellent tool for optical studies due to their innate smoothness and high quality factors, but precise control over their geometries can be difficult. In contrast, three dimensional (3D) printed components are highly customizable but suffer from roughness and pixelation. We present 3D printed structures which leverage the versatility of 3D printing with the smoothness of microdroplets. Our devices enable the reliable creation of microdroplet resonators of varying shapes and sizes in an ambient environment, and our coupling scheme allows for high control over droplet position.
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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.
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Dispositivos Laboratorio en un Chip , Nacimiento Prematuro , Biomarcadores , Humanos , Recién Nacido , Nacimiento Prematuro/diagnóstico , Impresión Tridimensional , SueroRESUMEN
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.
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A 3D printed, automated, pressure-driven injection microfluidic system for microchip electrophoresis (µCE) of preterm birth (PTB)-related peptides and proteins has been developed. Functional microvalves were formed, either with a membrane thickness of 5 µm and a layer exposure time of 450 ms or with a membrane thickness of 10 µm and layer exposure times of 300-350 ms. These valves allowed for control of fluid flow in device microchannels during sample injection for µCE separation. Device design and µCE conditions using fluorescently labeled amino acids were optimized. A sample injection time of 0.5 s and a separation voltage of 450 V (460 V/cm) yielded the best separation efficiency and resolution. We demonstrated the first µCE separation with pressure-driven injection in a 3D printed microfluidic device using fluorescently labeled PTB biomarkers and 532 nm laser excitation. Detection limits for two PTB biomarkers, peptide 1 and peptide 2, for an injection time of 1.5 s were 400 pM and 15 nM, respectively, and the linear detection range for peptide 2 was 50-400 nM. This 3D printed microfluidic system holds promise for future integration of on-chip sample preparation processes with µCE, offering promising possibilities for PTB risk assessment.
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Electroforesis por Microchip , Nacimiento Prematuro , Biomarcadores/análisis , Electroforesis por Microchip/métodos , Femenino , Humanos , Recién Nacido , Dispositivos Laboratorio en un Chip , Péptidos , Embarazo , Nacimiento Prematuro/diagnóstico , Impresión TridimensionalRESUMEN
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.
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Fluorescencia , Dispositivos Laboratorio en un Chip , Nacimiento Prematuro/diagnóstico , Impresión Tridimensional , Extracción en Fase Sólida , Biomarcadores/análisis , Colorantes Fluorescentes/química , HumanosRESUMEN
This work demonstrates for the first time the creation of microchip electrophoresis devices with â¼50 µm cross-sectional dimensions by stereolithographic 3D printing and their application in the analysis of medically significant biomarkers related to risk for preterm birth (PTB). We determined that device current was linear with applied potential up to 800 V (620 V/cm). We optimized device and separation conditions using fluorescently labeled amino acids as a model system and compared the performance in our 3D printed microfluidic devices to that in other device materials commonly used for microchip electrophoresis analysis. We demonstrated for the first time microchip electrophoresis in a 3D printed device of three PTB biomarkers, including peptides and a protein, with suitable separation characteristics. Limits of detection for microchip electrophoresis in 3D printed microfluidic devices were also determined for PTB biomarkers to be in the high picomolar to low nanomolar range.
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Electroforesis por Microchip , Dispositivos Laboratorio en un Chip , Nacimiento Prematuro/diagnóstico , Impresión Tridimensional , Aminoácidos/química , Biomarcadores/análisis , Femenino , Colorantes Fluorescentes/química , Humanos , EmbarazoRESUMEN
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.
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Dispositivos Laboratorio en un Chip , Embarazo/sangre , Nacimiento Prematuro , Impresión Tridimensional/instrumentación , Biomarcadores/sangre , Femenino , Humanos , Recién Nacido , PolimerizacionRESUMEN
Three-dimensional (3D) printing has generated considerable excitement in recent years regarding the extensive possibilities of this enabling technology. One area in which 3D printing has potential, not only for positive impact but also for substantial improvement, is microfluidics. To date many researchers have used 3D printers to make fluidic channels directed at point-of-care or lab-on-a-chip applications. Here, we look critically at the cross-sectional sizes of these 3D printed fluidic structures, classifying them as millifluidic (larger than 1 mm), sub-millifluidic (0.5-1.0 mm), large microfluidic (100-500 µm), or truly microfluidic (smaller than 100 µm). Additionally, we provide our prognosis for making 10-100-µm cross-section microfluidic features with custom-formulated resins and stereolithographic printers. Such 3D printed microfluidic devices for bioanalysis will accelerate research through designs that can be easily created and modified, allowing improved assays to be developed.
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Dispositivos Laboratorio en un Chip , Impresión Tridimensional , Sistemas de Atención de PuntoRESUMEN
Pneumatically actuated, non-elastomeric membrane valves fabricated from polymerized polyethylene glycol diacrylate (poly-PEGDA) have been characterized for temporal response, valve closure, and long-term durability. A ~100 ms valve opening time and a ~20 ms closure time offer valve operation as fast as 8 Hz with potential for further improvement. Comparison of circular and rectangular valve geometries indicates that the surface area for membrane interaction in the valve region is important for valve performance. After initial fabrication, the fluid pressure required to open a closed circular valve is ~50 kPa higher than the control pressure holding the valve closed. However, after ~1000 actuations to reconfigure polymer chains and increase elasticity in the membrane, the fluid pressure required to open a valve becomes the same as the control pressure holding the valve closed. After these initial conditioning actuations, poly-PEGDA valves show considerable robustness with no change in effective operation after 115,000 actuations. Such valves constructed from non-adsorptive poly-PEGDA could also find use as pumps, for application in small volume assays interfaced with biosensors or impedance detection, for example.
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Microdroplet resonators provide an excellent tool for optical studies of water, but water microdroplets are difficult to maintain outside a carefully controlled environment. We present a method for maintaining a water microdroplet resonator on a 3D-printed hydrophobic surface in an ambient environment. The droplet is maintained through a passive microfluidic system that supplies water to the droplet through a vertical channel at a rate equivalent to its evaporation. In this manner, we are able to create and passively maintain water microdroplet resonators with quality factors as high as 3×108.
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BACKGROUND: Preterm birth (PTB) is a leading cause of neonatal mortality, such that the need for a rapid and accurate assessment for PTB risk is critical. Here, we developed a 3D printed microfluidic system that integrated solid-phase extraction (SPE) and microchip electrophoresis (µCE) of PTB biomarkers, enabling the combination of biomarker enrichment and labeling with µCE separation and fluorescence detection. RESULTS: Reversed-phase SPE monoliths were photopolymerized in 3D printed devices. Microvalves in the device directed sample between the SPE monolith and the injection cross-channel in the serpentine µCE channel. Successful on-chip preconcentration, labeling and µCE separation of four PTB-related polypeptides were demonstrated in these integrated microfluidic devices. We further show the ability of these devices to handle complex sample matrices through the successful analysis of labeled PTB biomarkers spiked into maternal blood serum. The detection limit was 7 nM for the PTB biomarker, corticotropin releasing factor, in 3D printed SPE-µCE integrated devices. SIGNIFICANCE: This work represents the first successful demonstration of integration of SPE and µCE separation of disease-linked biomarkers in 3D printed microfluidic devices. These studies open up promising possibilities for rapid bioanalysis of medically relevant analytes.
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Electroforesis por Microchip , Nacimiento Prematuro , Femenino , Recién Nacido , Humanos , Electroforesis por Microchip/métodos , Nacimiento Prematuro/diagnóstico , Biomarcadores/análisis , Extracción en Fase Sólida/métodos , Dispositivos Laboratorio en un Chip , Impresión TridimensionalRESUMEN
In this work, we present a new 3D printing technique that enables the realization of native digital micro-mirror device (DMD) resolution in negative features of a 3D printed part without improving 3D printer hardware and demonstrate the fabrication of fully integrated, biocompatible isoporous membranes with pore sizes as small as 7 µm. We utilize this technique to construct a microfluidic device that mimics an established organ-on-a-chip configuration, including an integrated isoporous membrane. Two cell populations are seeded on either side of the membrane and imaged as a proof of concept for other organ-on-a-chip applications. These 3D printed isoporous membranes can be leveraged for a wide variety of other mechanical and biological applications, creating new possibilities for seamlessly integrated, 3D printed microfluidic devices.
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Dispositivos Laboratorio en un Chip , Impresión TridimensionalRESUMEN
Organs-on-a-chip, or OoCs, are microfluidic tissue culture devices with micro-scaled architectures that repeatedly achieve biomimicry of biological phenomena. They are well positioned to become the primary pre-clinical testing modality as they possess high translational value. Current methods of fabrication have facilitated the development of many custom OoCs that have generated promising results. However, the reliance on microfabrication and soft lithographic fabrication techniques has limited their prototyping turnover rate and scalability. Additive manufacturing, known commonly as 3D printing, shows promise to expedite this prototyping process, while also making fabrication easier and more reproducible. We briefly introduce common 3D printing modalities before identifying two sub-types of vat photopolymerization - stereolithography (SLA) and digital light processing (DLP) - as the most advantageous fabrication methods for the future of OoC development. We then outline the motivations for shifting to 3D printing, the requirements for 3D printed OoCs to be competitive with the current state of the art, and several considerations for achieving successful 3D printed OoC devices touching on design and fabrication techniques, including a survey of commercial and custom 3D printers and resins. In all, we aim to form a guide for the end-user to facilitate the in-house generation of 3D printed OoCs, along with the future translation of these important devices.
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Sistemas Microfisiológicos , Impresión Tridimensional , Estereolitografía , Microtecnología , Dispositivos Laboratorio en un ChipRESUMEN
New microfluidic lab-on-a-chip capabilities are enabled by broadening the toolkit of devices that can be created using microfabrication processes. For example, complex geometries made possible by 3D printing can be used to approach microfluidic design and application in new or enhanced ways. In this paper, we demonstrate three distinct designs for microfluidic one-way (check) valves that can be fabricated using digital light processing stereolithography (DLP-SLA) with a poly(ethylene glycol) diacrylate (PEGDA) resin, each with an internal volume of 5-10 nL. By mapping flow rate to pressure in both the forward and reverse directions, we compare the different designs and their operating characteristics. We also demonstrate pumps for each one-way valve design comprised of two one-way valves with a membrane valve displacement chamber between them. An advantage of such pumps is that they require a single pneumatic input instead of three as for conventional 3D-printed pumps. We also characterize the achievable flow rate as a function of the pneumatic control signal period. We show that such pumps can be used to create a single-stage diffusion mixer with significantly reduced pneumatic drive complexity.
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We employed digital light processing-stereolithography 3D printing to create microfluidic devices with different designs for microchip electrophoresis (µCE). Short or long straight channel, and two- or four-turn serpentine channel microfluidic devices with separation channel lengths of 1.3, 3.1, 3.0, and 4.7 cm, respectively, all with a cross injector design, were fabricated. We measured current as a function of time and voltage to determine a separation time window and conditions for the onset of Joule heating in these designs. Separations in these devices were evaluated by performing µCE and measuring theoretical plate counts for electric field strengths near and above the onset of Joule heating, with fluorescently labeled glycine and phenylalanine as model analytes. We further demonstrated µCE of peptides and proteins related to preterm birth risk, showing increased peak capacity and resolution compared to previous results with 3D printed microdevices. These results mark an important step forward in the use of 3D printed microfluidic devices for rapid bioanalysis by µCE.
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Electroforesis por Microchip , Nacimiento Prematuro , Recién Nacido , Femenino , Humanos , Nacimiento Prematuro/diagnóstico , Dispositivos Laboratorio en un Chip , Biomarcadores , Impresión TridimensionalRESUMEN
We demonstrate a method to effectively 3D print microfluidic devices with high-resolution features using a biocompatible resin based on avobenzone as the UV absorber. Our method relies on spectrally shaping the 3D printer source spectrum so that it is fully overlapped by avobenzone's absorption spectrum. Complete overlap is essential to effectively limit the optical penetration depth, which is required to achieve high out-of-plane resolution. We demonstrate the high resolution in practice by 3D printing 15 µm square pillars in a microfluidic chamber, where the pillars are separated by 7.7 µm and are printed with 5 µm layers. Furthermore, we show reliable membrane valves and pumps using the biocompatible resin. Valves are tested to 1,000,000 actuations with no observable degradation in performance. Finally, we create a concentration gradient generation (CG) component and utilize it in two device designs for cell chemotaxis studies. The first design relies on an external dual syringe pump to generate source and sink flows to supply the CG channel, while the second is a complete integrated device incorporating on-chip pumps, valves, and reservoirs. Both device types are seeded with adherent cells that are subjected to a chemoattractant CG, and both show clear evidence of chemotactic cellular migration. Moreover, the integrated device demonstrates cellular migration comparable to the external syringe pump device. This demonstration illustrates the effectiveness of our integrated chemotactic assay approach and high-resolution biocompatible resin 3D printing fabrication process. In addition, our 3D printing process has been tuned for rapid fabrication, as printing times for the two device designs are, respectively, 8 and 15 min.
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Microfluidic devices (MFDs) printed in 3-D geometry using digital light projection to polymerize monomers often have surfaces that are not as hydrophobic as MFDs made from polydimethylsiloxane. Droplet microfluidics in these types of devices are subject to droplet adhesion and aqueous spreading on less hydrophobic MFD surfaces. We have developed a post-processing technique using hydrophobic monomers that renders the surfaces of these devices much more hydrophobic. The technique is fast and easy, and involves flowing monomer without initiator into the channels and then exposing the entire device to UV light that generates radicals from the initiator molecules remaining in the original 3-D polymerization. After treatment the channels can be cleared and the surface is more hydrophobic, as evidenced by higher contact angles with aqueous droplets. We hypothesize that radicals generated near the previously printed surfaces initiate polymerization of the hydrophobic monomers on the surfaces without bulk polymerization extending into the channels. The most hydrophobic surfaces were produced by treatment with an alkyl acrylate and a fluorinated acrylate. This technique could be used for surface treatment with other types of monomers to impart unique characteristics to channels in MFDs.