3D printed porous membrane integrated devices to study the chemoattractant induced behavioural response of aquatic organisms.

Biological models with genetic similarities to humans are used for exploratory research to develop behavioral screening tools and understand sensory-motor interactions. Their small, often mm-sized appearance raises challenges in the straightforward quantification of their subtle behavioral responses and calls for new, customisable research tools. 3D printing provides an attractive approach for the manufacture of custom designs at low cost; however, challenges remain in the integration of functional materials like porous membranes. Nanoporous membranes have been integrated with resin exchange using purpose-designed resins by digital light projection 3D printing to yield functionally integrated devices using a simple, economical and semi-automated process. Here, the impact of the layer thickness and layer number on the porous properties - parameters unique for 3D printing - are investigated, showing decreases in mean pore diameter and porosity with increasing layer height and layer number. From the same resin formulation, materials with average pore size between 200 and 600 nm and porosity between 45% and 61% were printed. Membrane-integrated devices were used to study the chemoattractant induced behavioural response of zebrafish embryos and planarians, both demonstrating a predominant behavioral response towards the chemoattractant, spending >85% of experiment time in the attractant side of the observation chamber. The presented 3D printing method can be used for printing custom designed membrane-integrated devices using affordable 3D printers and enable fine-tuning of porous properties through adjustment of layer height and number. This accessible approach is expected to be adopted for applications including behavioural studies, early-stage pre-clinical drug discovery and (environmental) toxicology.

Chemical Trends in Sample Preparation for Nucleic Acid Amplification Testing (NAAT): A Review.

Nucleic acid amplification testing facilitates the detection of disease through specific genomic sequences and is attractive for point-of-need testing (PONT); in particular, the early detection of microorganisms can alert early response systems to protect the public and ecosystems from widespread outbreaks of biological threats, including infectious diseases. Prior to nucleic acid amplification and detection, extensive sample preparation techniques are required to free nucleic acids and extract them from the sample matrix. Sample preparation is critical to maximize the sensitivity and reliability of testing. As the enzymatic amplification reactions can be sensitive to inhibitors from the sample, as well as from chemicals used for lysis and extraction, avoiding inhibition is a significant challenge, particularly when minimising liquid handling steps is also desirable for the translation of the assay to a portable format for PONT. The reagents used in sample preparation for nucleic acid testing, covering lysis and NA extraction (binding, washing, and elution), are reviewed with a focus on their suitability for use in PONT.

Development of a pumpless acoustofluidic device for rapid food pathogen detection.

Mixing, homogenization, separation, and filtration are crucial processes in miniaturized analytical systems employed for in-vitro biological, environmental, and food analysis. However, in microfluidic systems achieving homogenization becomes more challenging due to the laminar flow conditions, which lack the turbulent flows typically used for mixing in traditional analytical systems. Here, we introduce an acoustofluidic platform that leverages an acoustic transducer to generate microvortex streaming, enabling effective homogenizing of food samples. To reduce reliance on external equipment, tubing, and pump, which is desirable for Point-of-Need testing, our pumpless platform employs a hydrophilic yarn capable of continuous wicking for sample perfusion. Following the homogenization process, the platform incorporates an array of micropillars for filtering out large particles from the samples. Additionally, the porous structure of the yarn provides a secondary screening mechanism. The resulting system is compact, and reliable, and was successfully applied to the detection of Escherichia coli (E. coli) in two different types of berries using quantitative polymerase chain reaction (qPCR). The platform demonstrated a detection limit of 5 CFU g-1, showcasing its effectiveness in rapid and sensitive pathogen detection.

3D printed integrated nanoporous membranes for electroextraction of DNA.

3D printing is established as an alternative microfabrication approach, and while printer resolution limits the direct 3D printing of pore features in the micron/submicron range, the use of nanoporous materials allows for the integration of porous membranes in 3D printed devices. Here, nanoporous membranes were formed by digital light projection (DLP) 3D printing using a polymerization-induced phase separation (PIPS) resin formulation. A functionally integrated device was fabricated using resin exchange following a simple, semi-automated manufacturing process. Printing of porous materials from a PIPS resin formulations based on polyethylene glycol diacrylate 250 as monomer was investigated by varying exposure time, photoinitiator concentration, and porogen content to yield materials with average pore size varying from 30-800 nm. Aiming for printing a size-mobility trap for electrophoretic extraction of deoxyribonucleic acid (DNA), conditions for printing materials with a mean pore size of 346 nm and 30 nm were selected for integration in a fluidic device using a resin exchange approach. Under optimized conditions (12.5 V for 20 min), cell concentrations as low as 103 cells per mL were detected following amplification of the extract by quantitative polymerase chain reaction (qPCR) at a Cq of 29. The efficacy of the size/mobility trap formed by the two membranes is demonstrated by detecting DNA concentrations equivalent to the input detected in the extract while removing 73% of the protein in the lysate. The DNA extraction yield was not statistically different from that obtained using a spin column, but manual handling and equipment needs were significantly reduced. This study demonstrates that nanoporous membranes with tailored properties can be integrated into fluidic devices using a simple manufacturing process based on resin exchange DLP. The process was used to manufacture a size-mobility trap and applied for the electroextraction and purification of DNA from E. coli lysate with reduced processing time, manual handling, and equipment needs compared with a commercially sourced DNA extraction kit. Combining manufacturability and portability with ease of use, the approach has demonstrated potential for manufacturing and using devices used in point-of-need testing for diagnostic nucleic acid amplification testing.

Closed-loop control systems for pumps used in portable analytical systems.

The demand for accurate control of the flowrate/pressure in chemical analytical systems has given rise to the adoption of mechatronic approaches in analytical instruments. A mechatronic device is a synergistic system which combines mechanical, electronic, computer and control components. In the development of portable analytical devices, considering the instrument as a mechatronic system can be useful to mitigate compromises made to decrease space, weight, or power consumption. Fluid handling is important for reliability, however, commonly utilized platforms such as syringe and peristaltic pumps are typically characterized by flow/pressure fluctuations and slow responses. Closed loop control systems have been used effectively to decrease the difference between desired and realized fluidic output. This review discusses the way control systems have been implemented for enhanced fluidic control, categorized by pump type. Advanced control strategies used to enhance the transient and the steady state responses are discussed, along with examples of their implementation in portable analytical systems. The review is concluded with the outlook that the challenge in adequately expressing the complexity and dynamics of the fluidic network as a mathematical model has yielded a trend towards the adoption of experimentally informed models and machine learning approaches.

Recent developments in digital light processing 3D-printing techniques for microfluidic analytical devices.

Digital light processing (DLP) 3D printing is rapidly advancing and has emerged as a powerful additive manufacturing approach to fabricate analytical microdevices. DLP 3D-printing utilizes a digital micromirror device to direct the projected light and photopolymerize a liquid resin, in a layer-by-layer approach. Advances in vat and lift design, projector technology, and resin composition, allow accurate fabrication of microchannel structures as small as 18 × 20 µm. This review describes the latest advances in DLP 3D-printing technology with respect to instrument set-up and resin formulation and highlights key efforts to fabricate microdevices targeting emerging (bio-)analytical chemistry applications, including colorimetric assays, extraction, and separation.

Novel Boundary Lubrication Mechanisms from Molecular Pillows of Lubricin Brush-Coated Graphene Oxide Nanosheets.

There are numerous biomedical applications where the interfacial shearing of surfaces can cause wear and friction, which can lead to a variety of medical complications such as inflammation, irritation, and even bacterial infection. We introduce a novel nanomaterial additive comprised of two-dimensional graphene oxide nanosheets (2D-NSCs) coated with lubricin (LUB) to reduce the amount of tribological stress in biomedical settings, particularly at low shear rates where boundary lubrication dominates. LUB is a glycoprotein found in the articular joints of mammals and has recently been discovered as an ocular surface boundary lubricant. The ability of LUB to self-assemble into a "telechelic" brush layer on a variety of surfaces was exploited here to coat the top and bottom surfaces of the ultrathin 2D-NSCs in solution, effectively creating a biopolymer-coated nanosheet. A reduction in friction of almost an order of magnitude was measured at a bioinspired interface. This reduction was maintained after repeated washing (5×), suggesting that the large aspect ratio of the 2D-NSCs facilitates effective lubrication even at diluted concentrations. Importantly, and unlike LUB-only treatment, the lubrication effect can be eliminated over 15 rinsing cycles, suggesting that the LUB-coated 2D-NSCs do not exhibit any binding interactions with the shearing surfaces. The effective lubricating properties of the 2D-NSCs combined with full reversibility through rinsing make the LUB-coated 2D-NSCs an intriguing candidate as a lubricant for biomedical applications.

Inexpensive portable capillary electrophoresis instrument for Monitoring Zinc(II) in remote areas.

A compact, inexpensive capillary electrophoresis instrument was developed for monitoring metal ions and evaluated for Zn(II) in remote contaminated locations in western Tasmania, Australia. The portable instrument, measuring 21 cm x 10 cm x 7 cm, was powered from the USB port of a laptop computer and built from off-the-shelf components costing ∼$1200 USD. Electrophoretic separations were conducted using a fused silica capillary (10-50 µm I.D.), applying 8.5 kV over capillaries ranging from 25 cm to 40 cm in length. The capillary inlet was connected with an electrically grounded cross-piece as flow-through injection interface. Automated fluidic management was achieved by controlling four mini peristaltic pumps and a solenoid valve. Detection was realised using a purpose-built visible LED absorption detector, optimised for the detection of Co(II), Cu(II) and Zn(II) after complexation with 4-(2-Pyridylazo) resorcinol (PAR). Limits of detection of sub-µM were obtained. The instrument was tested for continuous operation in the laboratory for up to 3 months, and relative standard deviations of <5.4% were found over 945 consecutive injections. In the field, the system was able to measure 106 samples within 11 h, the time it can be powered from the laptop computer. As Field measurement of Zn(II) in western Tasmania was demonstrated to show capability for on-site metal testing.

An improved nucleic acid sequence-based amplification method mediated by T4 gene 32 protein.

The uptake of Nucleic Acid Sequence-Based Amplification (NASBA) for point of care testing may be hindered by a complexity in the workflow due the requirement of a thermal denaturation step to initiate the cyclic isothermal amplification before the addition of the amplification enzymes. Despite reports of successful enhancement of other DNA and RNA amplification methods using DNA and RNA binding proteins, this has not been reported for NASBA. Here, three single-stranded binding proteins, RecA, Extreme Thermostable Single-stranded binding protein (ET SSB) and T4 gene gp32 protein (gp32), were incorporated in NASBA protocol and used for single pot, one-step NASBA at 41 °C. Indeed, all SSBs showed significantly improved amplifications compared with the 2-step process, but only gp32 showed no non-specific aberrant amplification, and slightly improved the time-to-positivity in comparison with the conventional NASBA. For synthetic HIV-1 RNA, gp32 was found to improve the time-to-positivity (ttp) by average of 13.6% of one-step NASBA and 6.7% of conventional NASBA for the detection of HIV-1 RNA, showing its potential for simplifying the workflow as desirable for point of care applications of NASBA.

3D printing for the integration of porous materials into miniaturised fluidic devices: A review.

Porous materials facilitate the efficient separation of chemicals and particulate matter by providing selectivity through structural and surface properties and are attractive as sorbent owing to their large surface area. This broad applicability of porous materials makes the integration of porous materials and microfluidic devices important in the development of more efficient, advanced separation platforms. Additive manufacturing approaches are fundamentally different to traditional manufacturing methods, providing unique opportunities in the fabrication of fluidic devices. The complementary 3D printing (3DP) methods are each accompanied by unique opportunities and limitations in terms of minimum channel size, scalability, functional integration and automation. This review focuses on the developments in the fabrication of 3DP miniaturised fluidic devices with integrated porous materials, focusing polymer-based methods including fused filament fabrication (FFF), inkjet 3D printing and digital light projection (DLP). The 3DP methods are compared based on resolution, scope for multimaterial printing and scalability for manufacturing. As opportunities for printing pores are limited by resolution, the focus is on approaches to incorporate materials with sub-micron pores to be used as membrane, sorbent or stationary phase in separation science using Post-Print, Print-Pause-Print and In-Print processes. Technical aspects analysing the efficiency of the fabrication process towards scalable manufacturing are combined with application aspects evaluating the separation and/or extraction performance. The review is concluded with an overview on achievements and opportunities for manufacturable 3D printed membrane/sorbent integrated fluidic devices.

Facile fabrication of micro-/nanostructured, superhydrophobic membranes with adjustable porosity by 3D printing.

Porous membranes with special wetting properties have attracted great interest due to their various functions and wide applications, including water filtration, selective oil/water separation and oil skimming. Special wetting properties such as superhydrophobicity can be achieved by controlling the surface chemistry as well as the surface topography of a substrate. Three-dimensional (3D) printing is a promising method for the fast and easy generation of various structures. The most common method for 3D printing of superhydrophobic materials is a two-step fabrication process: 3D printing of user-defined topographies, such as surface structures or bulk porosity, followed by a chemical post-processing with low-surface energy chemicals such as fluorinated silanes. Another common method is using a hydrophobic polymer ink to print intricate surface structures. However, the resolution of most common printers is not sufficient to produce nano-/microstructured textures, moreover, the resulting delicate surface micro- or nanostructures are very prone to abrasion. Herein, we report a simple approach for 3D printing of superhydrophobic micro-/nanoporous membranes in a single step, combining the required topography and chemistry. The bulk porosity of this material, which we term "Fluoropor", makes it insensitive to abrasion. To achieve this, a photocurable fluorinated resin is mixed with a porogen mixture and 3D printed using a stereolithography (SLA) printing process. This way, micro-/nanoporous membranes with superhydrophobic properties with static contact angles of 164° are fabricated. The pore size of the membranes can be adjusted from 30 nm to 300 nm by only changing the porogen ratio in the mixture. We show the applicability of the printed membranes for oil/water separation and the formation of Salvinia layers which are of great interest for drag reduction in maritime transportation and fouling prevention.

Sheathless Separation of Cyanobacterial Anabaena by Shape Using Viscoelastic Microfluidics.

Cyanobacteria have a wide range of impact on natural ecosystems, and have been recognized as potentially rich sources of pharmacological and structurally interesting secondary metabolites. To better understand the basic molecular processes and mechanisms that influence and regulate the growth (like length) of cyanobacteria, or connections between environment, genotype, and phenotype, it would be essential to separate shape-synchronized cyanobacterial cell populations with relatively uniform length and size. This work proposes a novel and efficient method to separate cyanobacterial Anabaena by shape (rod aspect ratio) using viscoelastic microfluidics in a straight channel with expansion-contraction cavity arrays (ECCA channel). The biocompatible viscoelastic solutions with dissolved polymer would induce a combined effect of inertial lift force, elastic force, and secondary drag force for Anabaena flowing in it. Therefore, Anabaena with different lengths reach different lateral equilibrium positions and flow out from different outlets. Factors including flow rate, fluid viscoelasticity, channel structure, and length on the shape-based cell separation were studied systematically. This work, for the first time, demonstrates continuous and sheathless shape-based separation of cyanobacteria using viscoelastic microfluidics. Moreover, its ability to manipulate objects with different morphologies and with a size of >100 µm will extend the capability of microfluidics to a completely new field that has never been reached and would be attractive across a range of new applications.

Hyphenated sample preparation-electrospray and nano-electrospray ionization mass spectrometry for biofluid analysis.

Stand-alone electrospray ionization mass spectrometry (ESI-MS) has been advancing through enhancements in throughput, selectivity and sensitivity of mass spectrometers. Unlike traditional MS techniques which usually require extensive offline sample preparation and chromatographic separation, many sample preparation techniques are now directly coupled with stand-alone MS to enable outstanding throughput for bioanalysis. In this review, we summarize the different sample clean-up and/or analyte enrichment strategies that can be directly coupled with ESI-MS and nano-ESI-MS for the analysis of biological fluids. The overview covers the hyphenation of different sample preparation techniques including solid phase extraction (SPE), solid phase micro-extraction (SPME), slug flow micro-extraction/nano-extraction (SFME/SFNE), liquid extraction surface analysis (LESA), extraction electrospray, extraction using digital microfluidics (DMF), and electrokinetic extraction (EkE) with ESI-MS and nano-ESI-MS.

Antifouling Strategies for Electrochemical Biosensing: Mechanisms and Performance toward Point of Care Based Diagnostic Applications.

Although there exist numerous established laboratory-based technologies for sample diagnostics and analyte detection, many medical and forensic science applications require point of care based platforms for rapid on-the-spot sample analysis. Electrochemical biosensors provide a promising avenue for such applications due to the portability and functional simplicity of the technology. However, the ability to develop such platforms with the high sensitivity and selectivity required for analysis of low analyte concentrations in complex biological samples remains a paramount issue in the field of biosensing. Nonspecific adsorption, or fouling, at the electrode interface via the innumerable biomolecules present in these sample types (i.e., serum, urine, blood/plasma, and saliva) can drastically obstruct electrochemical performance, increasing background "noise" and diminishing both the electrochemical signal magnitude and specificity of the biosensor. Consequently, this review aims to discuss strategies and concepts used throughout the literature to prevent electrode surface fouling in biosensors and to communicate the nature of the antifouling mechanisms by which they operate. Evaluation of each antifouling strategy is focused primarily on the fabrication method, experimental technique, sample composition, and electrochemical performance of each technology highlighting the overall feasibility of the platform for point of care based diagnostic/detection applications.

Scalable 3D printing method for the manufacture of single-material fluidic devices with integrated filter for point of collection colourimetric analysis.

Assembly and bonding are major obstacles in manufacturing of functionally integrated fluidic devices. Here we demonstrate a single-material 3D printed device with an integrated porous structure capable of filtering particulate matter for the colourimetric detection of iron from soil and natural waters. Selecting a PolyJet 3D printer for its throughput, integrated filters were created exploiting a phenomenon occurring at the interface between the commercially available build material (Veroclear-RGD810) and water-soluble support material (SUP707). The porous properties were tuneable by varying the orientation of the print head relative to the channel and by varying the width of the build material. Porous structures ranging from 100 to 200 µm in thickness separated the sample and reagent chambers, filtering particles larger than 15 µm in diameter. Maintaining the manufacturing throughput of the Polyjet printer, 221 devices could be printed in 1.5 h (∼25 s per device). Including the 12 h post-processing soak in sodium hydroxide to remove the solid support material, the total time to print and process 221 devices was 13.5 h (3.6 min per device), with a material cost of $2.50 each. The applicability of the fluidic device for point of collection analysis was evaluated using colourimetric determination of iron from soil slurry and environmental samples. Following the reduction of Fe3+ to Fe2+ using hydroxylammonium chloride, samples were introduced to the fluidic device where particulate matter was retained by the filter, allowing for particulate-free imaging of the red complex formed with 1,10-phenanthroline using a smartphone camera. The calibration curve ranged from of 1-100 mg L-1 Fe2+ and good agreement (95%) was obtained between the point of collection device and Sector Field ICP-MS.

3D Printing: An Alternative Microfabrication Approach with Unprecedented Opportunities in Design.

In the past decade, 3D printing technologies have been adopted for the fabrication of microfluidic devices. Extrusion-based approaches including fused filament fabrication (FFF), jetting technologies including inkjet 3D printing, and vat photopolymerization techniques including stereolithography (SLA) and digital light projection (DLP) are the 3D printing methods most frequently adopted by the microfluidic community. Each printing technique has merits toward the fabrication of microfluidic devices. Inkjet printing offers a good selection of materials and multimaterial printing, and the large build space provides manufacturing throughput, while FFF offers a great selection of materials and multimaterial printing but at lower throughput compared to inkjet 3D printing. Technical and material developments adopted from adjacent research fields and developed by the microfluidic community underpin the printing of sub-100 µm enclosed microchannels by DLP, but challenges remain in multimaterial printing throughput. With the feasibility of 3D printed microfluidics established, we look ahead at trends in 3D printing to gain insights toward the future of this technology beyond the sole prism of being an alternative fabrication approach. A shift in emphasis from using 3D printing for prototyping, to mimic conventionally manufactured outputs, toward integrated approaches from a design perspective is critically developed.

In-Syringe Electrokinetic Protein Removal from Biological Samples prior to Electrospray Ionization Mass Spectrometry.

Here, an electrokinetic extraction (EkE) syringe is presented allowing for on-line electrokinetic removal of serum proteins before ESI-MS. The proposed concept is demonstrated by the determination of pharmaceuticals from human serum within minutes, with sample preparation limited to a 5× dilution of the sample in the background electrolyte (BGE) and application of voltage, both of which can be performed in-syringe. Signal enhancements of 3.6-32 fold relative to direct infusion of diluted serum and up to 10.8 fold enhancement, were obtained for basic and acidic pharmaceuticals, respectively. Linear correlations for the basic drugs by EkE-ESI-MS/MS were achieved, covering the necessary clinical range with LOQs of 5.3, 7.8, 6.1, and 17.8 ng mL-1 for clomipramine, chlorphenamine, pindolol, and atenolol, respectively. For the acidic drugs, the EkE-ESI-MS LOQs were 3.1 µg mL-1 and 2.9 µg mL-1 for naproxen and paracetamol, respectively. The EkE-ESI-MS and EkE-ESI-MS/MS methods showed good accuracy (%found of 81 % to 120 %), precision (≤20 %), and linearity (r>0.997) for all the studied drugs in spiked serum samples.

The influence of electrolyte concentration on nanofractures fabricated in a 3D-printed microfluidic device by controlled dielectric breakdown.

A three-dimensional-printed microfluidic device made of a thermoplastic material was used to study the creation of molecular filters by controlled dielectric breakdown. The device was made from acrylonitrile butadiene styrene by a fused deposition modeling three-dimensional printer and consisted of two V-shaped sample compartments separated by 750 µm of extruded plastic gap. Nanofractures were formed in the thin piece of acrylonitrile butadiene styrene by controlled dielectric breakdown by application voltage of 15-20 kV with the voltage terminated when reaching a defined current threshold. Variation of the size of the nanofractures was achieved by both variation of the current threshold and by variation of the ionic strength of the electrolyte used for breakdown. Electrophoretic transport of two proteins, R-phycoerythrin (RPE; <10 nm in size) and fluorescamine-labeled BSA (f-BSA; 2-4 nm), was used to monitor the size and transport properties of the nanofractures. Using 1 mM phosphate buffer, both RPE and f-BSA passed through the nanofractures when the current threshold was set to 25 µA. However, when the threshold was lowered to 10 µA or lower, RPE was restricted from moving through the nanofractures. When we increased the electrolyte concentration during breakdown from 1 to 10 mM phosphate buffer, BSA passed but RPE was blocked when the threshold was equal to, or lower than, 25 µA. This demonstrates that nanofracture size (pore area) is directly related to the breakdown current threshold but inversely related to the concentration of the electrolyte used for the breakdown process.

One step multi-material 3D printing for the fabrication of a photometric detector flow cell.

Optical detection is the most common detection mode for many analytical assays. Photometric detection systems and their integration with analytical systems usually require several assembly parts and manual alignment of the capillary/tubing which affects sensitivity and repeatability. 3D printing is an innovative technology for the fabrication of integrated complex detection systems. One step multi-material 3D printing has been explored to fabricate a photometric detector flow cell from optically transparent and opaque materials using a dual-head FDM 3D printer. Integration of the microchannel, the detection window and the slit in a single device eliminates the need for manual alignment of fluidic and optical components, and hence improves sensitivity and repeatability. 3D printing allowed for rapid design optimisation by varying the slit dimension and optical pathlength. The optimised design was evaluated by determining stray light, effective path length and the signal to noise ratio using orange G. The optimised flow cell with extended path length of 10 mm and 500 µm slit yielded 0.02% stray light, 89% effective path length and detection limit of 2 nM. The sensitivity was also improved by 80% in the process of optimisation, using a blue 470 nm LED as a light source.

Rapid Additive Manufacturing of 3D Geometric Structures via Dual-Wavelength Polymerization.

A dual-wavelength photopolymerization process is presented, allowing for the volumetric fabrication of complex geometries using a multistep process. The methacrylate-based resin contained 0.1 wt % camphorquinone/0.1 wt % ethyl 4-(dimethylamino) benzoate and 0.2 wt % bis[2-(ochlorophenyl)-4,5-diphenylimidazole] as photoinitiator (473 nm) and photoinhibitor (365 nm), respectively. The photoinitiator and photoinhibitor concentrations were optimized to allow for photocuring to full depth (4.6 mm) following an exposure time of 2 min solely by 473 nm light, but no curing occurred when 365 nm light was present due to photoinhibition. This resin was validated using one-step volumetric fabrication of two objects containing voids defined by the 365 nm irradiation region. Two more complex structures were printed in a step-by-step manner, relying on the dynamic control of the projection patterns of both 365 and 473 nm projectors, decreasing the print time from 20 min using a commercially available single wavelength resin printer to 2 min.