3D-printed microchip electrophoresis device containing spiral electrodes for integrated capacitively coupled contactless conductivity detection.

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.

3D Printed Microfluidics.

Traditional microfabrication techniques suffer from several disadvantages, including the inability to create truly three-dimensional (3D) architectures, expensive and time-consuming processes when changing device designs, and difficulty in transitioning from prototyping fabrication to bulk manufacturing. 3D printing is an emerging technique that could overcome these disadvantages. While most 3D printed fluidic devices and features to date have been on the millifluidic size scale, some truly microfluidic devices have been shown. Currently, stereolithography is the most promising approach for routine creation of microfluidic structures, but several approaches under development also have potential. Microfluidic 3D printing is still in an early stage, similar to where polydimethylsiloxane was two decades ago. With additional work to advance printer hardware and software control, expand and improve resin and printing material selections, and realize additional applications for 3D printed devices, we foresee 3D printing becoming the dominant microfluidic fabrication method.

Physiological resistance alters behavioral response of Tetranychus urticae to acaricides.

Multiple acaricide resistance in Tetranychus urticae continues to threaten crop production globally, justifying the need to adequately study resistance for sustainable pest management. Most studies on acaricide resistance have focused on the acute contact toxicity of acaricides with little or no information on the behavioral responses elicited after acaricide exposure. Furthermore, the impact of physiological resistance on these behavioral responses remains unknown in most pest species, including T. urticae. We tested the effect of acaricide resistance on contact toxicity, irritancy and repellency of mitochondrial electron transport inhibitor of complex I (MET-I) and mite growth inhibitor (MGI) acaricides on multiple T. urticae strains. We also tested whether acaricides with similar physiological target site/mode of action also elicit similar behavioral effects on T. urticae strains. MET-I acaricides (fenazaquin, fenpyroximate, and pyrabiden) and MGIs (clofentezine, hexythiazox and etoxazole) elicited a dose-dependent irritant and repellent effect on T. urticae. Selection of strains for physiological resistance to these acaricides affected the behavioral response of T. urticae, especially in MET-I resistant strains, that showed reduced irritancy and repellency to MET-I acaricides. Behavioral response also affected the oviposition of T. urticae, where strains generally showed preferential oviposition away from the acaricides. The outcome of this study highlights negative consequences of acaricide resistance that can potentially affect T. urticae management.

3D Printed Microfluidic Devices for Microchip Electrophoresis of Preterm Birth Biomarkers.

This work demonstrates for the first time the creation of microchip electrophoresis devices with ∼50 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.

3D printed microfluidic devices with immunoaffinity monoliths for extraction of preterm birth biomarkers.

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.

3D Printed Microfluidic Features Using Dose Control in X, Y, and Z Dimensions.

Interest has grown in recent years to leverage the possibilities offered by three-dimensional (3D) printing, such as rapid iterative changes; the ability to more fully use 3D device volume; and ease of fabrication, especially as it relates to the creation of complex microfluidic devices. A major shortcoming of most commercially available 3D printers is that their resolution is not sufficient to produce features that are truly microfluidic (<100 × 100 µm²). Here, we test a custom 3D printer for making ~30 µm scale positive and negative surface features, as well as positive and negative features within internal voids (i.e., microfluidic channels). We found that optical dosage control is essential for creating the smallest microfluidic features (~30 µm wide for ridges, ~20 µm wide for trenches), and that this resolution was achieved for a number of different exposure approaches. Additionally, we printed various microfluidic particle traps, showed capture of 25 µm diameter polymer beads, and iteratively improved the trap design. The rapid feedback allowed by 3D printing, as well as the ability to carefully control optical exposure conditions, should lead to new innovations in the types and sizes of devices that can be created for microfluidics.

Moving from millifluidic to truly microfluidic sub-100-µm cross-section 3D printed devices.

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.

Reciprocal regulation of beta(1)-adrenergic receptor gene transcription by Sp1 and early growth response gene 1: induction of EGR-1 inhibits the expression of the beta(1)-adrenergic receptor gene.

The beta(1)-adrenergic receptor (beta(1)-AR) plays a key role in regulating heart rate and contractility in response to catecholamines. Our studies have focused on defining the factors that regulate the expression of the beta(1)-AR gene. We determined that a 65-base-pair (bp) region in the beta(1)-AR promoter between bp -394 and bp -330 directs basal transcription. An element located between -377 and -365 can bind Sp1 and Sp3. In Drosophila melanogaster SL2 cells, Sp1 stimulated the expression of the beta(1)-AR promoter, whereas Sp3 was unable to activate transcription. Site-directed mutagenesis indicated that an intact Sp1-binding site is essential for maintaining the activity of the basal promoter. In addition to binding Sp family members, the nucleotides between -381 and -367 can bind the zinc-finger transcription factor Egr-1. The Egr-1 and Sp1 binding sites are partially overlapping and their binding sequence is conserved among mammalian beta(1)-AR genes. The induction of Egr-1 in rat neonatal ventricular myocytes with phorbol-12-myristate-13-acetate or in HeLa S3 cells by regulated expression of Egr-1 in a tetracycline-responsive promoter, suppressed expression from the beta(1)-AR promoter. Overexpression of Sp1 in SK-N-MC cells increased beta(1)-AR mRNA by 2.4-fold, whereas overexpression of Egr-1 reduced beta(1)-AR mRNA by 40%. Coexpression of Egr-1 with Sp1 reduced Sp1-mediated up-regulation of beta(1)-AR mRNA by 60%. Mutagenesis revealed that an intact Sp1-binding site is essential for observing transcriptional repression by Egr-1 and that Egr-1 suppressed the transcription of the beta(1)-AR gene by competing with Sp1 for binding to their overlapping sites. These results reveal a novel physiologically relevant transcriptional mechanism for reciprocal regulation of beta(1)-AR gene expression.