Increased demand for inexpensive and rapid prototyping methods for micro- and millifluidic lab-on-a-chip (LOC) devices has stimulated considerable interest in alternative cost-effective fabrication techniques. Additive manufacturing (AM)—also called three-dimensional (3D) printing—provides an attractive alternative to conventional fabrication techniques. AM has been used to produce LOC master moulds from which positive replicas are made using soft-lithography and a biocompatible elastomer, poly(dimethylsiloxane) (PDMS). Here we characterize moulds made using two AM methods—stereolithography (SLA) and material-jetting (MJ)—and the positive replicas produced by soft lithography and PDMS moulding. The results showed that SLA, more than MJ, produced finer part resolution and finer tuning of feature geometry. Furthermore, as assessed by zebrafish (Danio rerio) biotoxicity tests, there was no toxicity observed in SLA and MJ moulded PDMS replicas. We conclude that SLA, utilizing commercially available printers and resins, combined with PDMS soft-lithography, is a simple and easily accessible technique that lends its self particularly well to the fabrication of biocompatible millifluidic devices, highly suited to the in-situ analysis of small model organisms.

Dechorionation of zebrafish embryos has been proposed as a tool to improve toxicity bioassays because the chorion membrane forms a molecular barrier that can slow down or prevent chemicals from reaching the embryo. Despite its potential importance for experimental and predictive toxicology, the culture of dechorionated zebrafish embryos in a microfluidic environment has so far not received any attention. Here, we demonstrate a new Lab-on-a-Chip technology capable of automated, hydrodynamic immobilization of dechorionated embryos of Danio rerio. We show that chorion-less embryos can develop normally under microfluidic perfusion and be successfully used for on-chip developmental toxicity bioassays.

At present, the major hurdle to widespread deployment of zebrafish embryo and larvae in large-scale drug development projects is lack of enabling high-throughput analytical platforms. In order to spearhead drug discovery with the use of zebrafish as a model, platforms need to integrate automated pre-test sorting of organisms (to ensure quality control and standardization) and their in-test positioning (suitable for high-content imaging) with modules for flexible drug delivery. The major obstacle hampering sorting of millimetre sized particles such as zebrafish embryos on chip-based devices is their substantial diameter (above one millimetre), mass (above one milligram), which both lead to rapid gravitational-induced sedimentation and high inertial forces. Manual procedures associated with sorting hundreds of embryos are very monotonous and as such prone to significant analytical errors due to operator's fatigue. In this work, we present an innovative design of a micromechanical large particle in-flow sorter (MILPIS) capable of analysing, sorting and dispensing living zebrafish embryos for drug discovery applications. The system consisted of a microfluidic network, revolving micromechanical receptacle actuated by robotic servomotor and opto-electronic sensing module. The prototypes were fabricated in poly(methyl methacrylate) (PMMA) transparent thermoplastic using infrared laser micromachining. Elements of MILPIS were also fabricated in an optically transparent VisiJet resin using 3D stereolithography (SLA) processes (ProJet 7000HD, 3D Systems). The device operation was based on a rapidly revolving miniaturized mechanical receptacle. The latter function was to hold and position individual fish embryos for (i) interrogation, (ii) sorting decision-making and (iii) physical sorting..The system was designed to separate between fertilized (LIVE) and non-fertilized (DEAD) eggs, based on optical transparency using infrared (IR) emitters and receivers embedded in the system. Digital oscilloscope were used to distinguish the diffraction signals from IR sensors when both LIVE and DEAD embryos were flow through in the chip. Image process analysis were also used as detection module to track DEAD embryos as it flowed in the channel. ; phone 61 3 992 57157; fax 61 3 992 57110; http://www.rmit.edu.au/staff/donald-wlodkowic

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