PolyJet 3D-Printed Enclosed Microfluidic Channels without Photocurable Supports.

Microfluidic devices have historically been prepared using fabrication techniques that often include photolithography and/or etching. Recently, additive manufacturing technologies, commonly known as 3D-printing, have emerged as fabrication tools for microfluidic devices. Unfortunately, PolyJet 3D-printing, which utilizes a photocurable resin that can be accurately printed, requires the use of support material for any designed void space internal to the model. Removing the support material from the printed channels is difficult in small channels with single dimensions of less than ∼200 µm and nearly impossible to remove from designs that contain turns or serpentines. Here, we describe techniques for printing channels ranging in cross sections from 0.6 cm × 1.5 cm to 125 µm × 54 µm utilizing commercially available PolyJet printers that require minimal to no postprocessing to form sealed channels. Specifically, printer software manipulation allows printing of one model with an open channel or void that is sealed with either a viscous liquid or a polycarbonate membrane (no commercially available support material). The printer stage is then adjusted and a second model is printed directly on top of the first model with the selected support system. Both the liquid-fill and the membrane method have enough structural integrity to support the printing resin while it is being cured. Importantly, such complex channel geometries as serpentine and Y-mixers can be designed, printed, and in use in under 2 h. We demonstrate device utility by measuring ATP release from flowing red blood cells using a luciferin/luciferase chemiluminescent assay that involves on-chip mixing and optical detection.

Ultrafiltration binding analyses of glycated albumin with a 3D-printed syringe attachment.

Protein-ligand binding assays facilitate the understanding of biomolecular interactions. Classical equilibrium dialysis methods are often used for accurate determination of binding properties. While accurate, the long equilibration times associated with the technique (> 6 h) hinder throughput. Here, in an attempt to gather high-accuracy results while reducing total analysis time, a low pressure ultrafiltration method that relies on a simple membrane-containing syringe attachment was developed. A minimal portion (1-2%) of the solution containing the binding analytes of interest is driven through the membrane pores and collected for analysis. Specifically, the device was used to investigate the binding affinity between Zn2+ and either normal human serum albumin (nHSA) or a commercially purchased glycated human serum albumin (gHSA). Both of these ligand/protein-binding systems have implications in type 1 diabetes. The device was then used to investigate the binding between the various albumin types and C-peptide, the 31 amino acid peptide that is co-secreted with insulin from pancreatic ß cells. Results for nHSA/Zn2+ binding obtained using the ultrafiltration method (Kd = 5.77 ± 0.19 × 10-7 M) were statistically equivalent with results reported using other methods. Importantly, the amount of Zn2+ bound to the nHSA was significantly different from the gHSA (97 ± 2% protein bound vs. 91 ± 3%, respectively p < 0.05). The binding affinity of C-peptide to nHSA (Kd = 2.4 ± 0.3 × 10-6 M) agreed with values reported in the literature using standard techniques. Unlike Zn2+ binding, the binding of C-peptide to nHSA was statistically equal to its binding to gHSA (77.7 ± 6.2 and 78.8 ± 7.4%, respectively), suggesting that C-peptide replacement therapy in people with T1D may be strongly dependent upon the characteristics of Zn2+ binding to human serum albumin. Graphical abstract ᅟ.

A Printed Equilibrium Dialysis Device with Integrated Membranes for Improved Binding Affinity Measurements.

Equilibrium dialysis is a simple and effective technique used for investigating the binding of small molecules and ions to proteins. A three-dimensional (3D) printer was used to create a device capable of measuring binding constants between a protein and a small ion based on equilibrium dialysis. Specifically, the technology described here enables the user to customize an equilibrium dialysis device to fit their own experiments by choosing membranes of various material and molecular-weight cutoff values. The device has dimensions similar to that of a standard 96-well plate, thus being amenable to automated sample handlers and multichannel pipettes. The device consists of a printed base that hosts multiple windows containing a porous regenerated-cellulose membrane with a molecular-weight cutoff of ∼3500 Da. A key step in the fabrication process is a print-pause-print approach for integrating membranes directly into the windows subsequently inserted into the base. The integrated membranes display no leaking upon placement into the base. After characterizing the system's requirements for reaching equilibrium, the device was used to successfully measure an equilibrium dissociation constant for Zn2+ and human serum albumin (Kd = (5.62 ± 0.93) × 10-7 M) under physiological conditions that is statistically equal to the constants reported in the literature.

Assessing methods for detecting Alexandrium catenella (Dinophyceae) and paralytic shellfish toxins in Southeast Alaska.

Blooms of Alexandrium catenella threaten to disrupt subsistence, recreational, and commercial shellfish harvest in Alaska, as the paralytic shellfish toxins (PSTs) produced pose a serious public health risk and can lead to costly shutdowns for shellfish farmers. Current methods of PST detection in the region range from monitoring programs utilizing net tows to detect A. catenella to direct shellfish tissue testing via mouse bioassay (MBA) for commercial aquaculture harvest, as well as various optional testing methods for subsistence and recreational harvesters. The efficacy and feasibility of these methods vary, and they have not been directly compared in Southeast Alaska. In this study, we sought to assess and compare A. catenella and PST early detection methods to determine which can provide the most effective and accurate warning of A. catenella blooms or PST events. We found microscope counts to be variable and prone to missing lower numbers of A. catenella, which may be indicative of bloom formation. However, quantitative polymerase chain reaction (qPCR) significantly correlated with microscope counts and was able to effectively detect even low numbers of A. catenella on all sampling days. Paralytic shellfish toxin concentrations measured by enzyme-linked immunosorbent assay and MBA significantly correlated with each other, qPCR, and some microscope counts. These results show that qPCR is an effective tool for both monitoring A. catenella and serving as a proxy for PSTs. Further work is needed to refine qPCR protocols in this system to provide bloom warnings on an actionable timescale for the aquaculture industry and other shellfish harvesters. Integr Environ Assess Manag 2024;00:1-14. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

A novel 3D-printed centrifugal ultrafiltration method reveals in vivo glycation of human serum albumin decreases its binding affinity for zinc.

Plasma proteins are covalently modified in vivo by the high-glucose conditions in the bloodstreams of people with diabetes, resulting in changes to both structure and function. Human Serum Albumin (HSA) functions as a carrier-protein in the bloodstream, binding various ligands and tightly regulating their bioavailability. HSA is known to react with glucose via the Maillard reaction, causing adverse effects on its ability to bind and deliver certain ligands, such as metals. Here, the binding between in vivo glycated HSA and zinc (Zn2+) was determined using a novel centrifugal ultrafiltration method that was developed using a 3D-printed device. This method is rapid (90 minutes), capable of high-throughput measurements (24 samples), low-cost (<$1.00 USD per device) and requires lower sample volumes (200 µL) compared to other binding techniques. This device was used to determine an equilibrium dissociation constant between Zn2+ and a commercially obtained normal HSA (nHSA) with a glycation level of 11.5% (Kd = 2.1 (±0.5) × 10-7 M). A glycated fraction of the nHSA sample was enriched (gHSA, 65.5%) and isolated using boronate-affinity chromatography, and found to have a 2.3-fold decrease in Zn2+ binding-affinity (Kd = 4.8 (±0.8) × 10-7 M) when compared to the nHSA sample. The level of glycation of HSA in control plasma (13.0% ± 0.8, n = 3 donors) and plasma from people with diabetes (26.9% ± 6.6, n = 5 donors) was assessed using mass spectrometry. Furthermore, HSA was isolated from plasma obtained in-house from a person with type 1 diabetes and found to have a glycation level of 24.1% and Kd = 3.3 (± 0.5) × 10-7 M for Zn2+, revealing a 1.5-fold decrease in binding affinity compared to nHSA. These findings suggest that increased levels of glycated HSA result in reduced binding to Zn2+, which may have implications in complications associated with diabetes.