Improving the limit of detection in portable luminescent assay readers through smart optical design

Critical biomarkers of disease are increasingly being detected by point‐of‐care assays. Chemiluminescence (CL) and electrochemiluminescence (ECL) are often used in such assays due to their convenience and that they do not require light sources or other components that could complicate or add cost to the system. Reports of these assays often include readers built on a cellphone platform or constructed from low‐cost components. However, the impact the optical design has on the limit of detection (LOD) in these systems remains unexamined. Here, we report a theoretical rubric to evaluate different optical designs in terms of maximizing the use of photons emitted from a CL or ECL assay to improve the LOD. We demonstrate that the majority of cellphone designs reported in the literature are not optimized, in part due to misunderstandings of the optical tradeoffs in collection systems, and in part due to limitations imposed on the designs arising from the use of a mobile phone with a very small lens aperture. Based on the theoretical rubric, we design a new portable reader built using off‐the‐shelf condenser optics, and demonstrate a nearly 10× performance enhancement compared to prior reports on an ECL assays running on a portable chip.     

Open-Source Syringe Pump Library

This article explores a new open-source method for developing and manufacturing high-quality scientific equipment suitable for use in virtually any laboratory. A syringe pump was designed using freely available open-source computer aided design (CAD) software and manufactured using an open-source RepRap 3-D printer and readily available parts. The design, bill of materials and assembly instructions are globally available to anyone wishing to use them. Details are provided covering the use of the CAD software and the RepRap 3-D printer. The use of an open-source Rasberry Pi computer as a wireless control device is also illustrated. Performance of the syringe pump was assessed and the methods used for assessment are detailed. The cost of the entire system, including the controller and web-based control interface, is on the order of 5% or less than one would expect to pay for a commercial syringe pump having similar performance. The design should suit the needs of a given research activity requiring a syringe pump including carefully controlled dosing of reagents, pharmaceuticals, and delivery of viscous 3-D printer media among other applications.     

Combined Ring–Mesh Optical Transport Networks

To date in Sonet and DWDM transport environments, we see two dominant principles for network survivability: ring and mesh. Although both are used, they are virtually always deployed and operated on geographically separate networks or regions, or applied on a ring-access and mesh-core principle. We describe a new approach for optimized design of ring and mesh network components in a single hybrid transport network. A key concept in design of these hybrids is the clipping of forcer spans in the mesh network design by strategically added rings. We present a formal optimization model and a heuristic to form sych hybrid designs with lower total cost than a pure-mesh design. These ideas on optical network architecture are especially relevant to industry practice with the advent of OXC systems with integrated add–drop multiplexer functionality and by the development of ultra long reach (ULR) optics. This integration of OXC-OADM functionality effectively removes any extra costs associated with transitions from a ring to mesh environment and ULR optics permit all-optical bypass of OXC nodes which enhances the cost-effectiveness of the hybrid architectures.     

Flow cytometers using optical waveguides in place of lenses for specimen illumination and light collection

Optical waveguides represent alternatives to lenses for delivery of light to, and collection of light from, small regions of space such as the observation point(s) of a flow cytometer. The flow chamber and directly associated illumination and collection optics of a flow cytometer can be built using fiber optic waveguides in fixed positions or as an integrated optical assembly incorporating deposited or implanted waveguides, providing illumination and collection geometries and efficiencies comparable to conventional designs, with the advantage that the optical waveguide design, once assembled, never again requires alignment of the optics. A prototype apparatus in which fiber optics were used to illuminate and collect light from a point inside a thin-walled, round quartz capillary was observed to measure fluorescence of polystyrene spheres with precision close to that obtained from a conventional flow cytometer using the same laser source. The design of the fiber optic system is readily adaptable to construction of multiple illumination beam instruments; other optical waveguide flow cytometer designs that dispense with the capillary share this advantage and can also provide orifices for electronic volume sensing and for jetting prior to droplet sorting.     

Micro-optical artificial compound eyes

Natural compound eyes combine small eye volumes with a large field of view at the cost of comparatively low spatial resolution. For small invertebrates such as flies or moths, compound eyes are the perfectly adapted solution to obtaining sufficient visual information about their environment without overloading their brains with the necessary image processing. However, to date little effort has been made to adopt this principle in optics. Classical imaging always had its archetype in natural single aperture eyes which, for example, human vision is based on. But a high-resolution image is not always required. Often the focus is on very compact, robust and cheap vision systems. The main question is consequently: what is the better approach for extremely miniaturized imaging systems-just scaling of classical lens designs or being inspired by alternative imaging principles evolved by nature in the case of small insects? In this paper, it is shown that such optical systems can be achieved using state-of-the-art micro-optics technology. This enables the generation of highly precise and uniform microlens arrays and their accurate alignment to the subsequent optics-, spacing- and optoelectronics structures. The results are thin, simple and monolithic imaging devices with a high accuracy of photolithography. Two different artificial compound eye concepts for compact vision systems have been investigated in detail: the artificial apposition compound eye and the cluster eye. Novel optical design methods and characterization tools were developed to allow the layout and experimental testing of the planar micro-optical imaging systems, which were fabricated for the first time by micro-optics technology. The artificial apposition compound eye can be considered as a simple imaging optical sensor while the cluster eye is capable of becoming a valid alternative to classical bulk objectives but is much more complex than the first system.     

Design and fabrication of a silica on silicon integrated optical biochip as a fluorescence microarray platform

Previous research into the use of Flame Hydrolysis Deposition (FHD) of glasses in integrated optics has focused on the successful commercial exploitation of low cost optical devices within the field of telecommunications and optoelectronics. Recently we have sought to apply these fabrication technologies to the development of optical biochips, utilising their ability to be integrated with microfluidics as a 'Lab-on-a-chip' platform. In this paper, we carry this development forward by seeking to create a microarray of integrated optical sensing elements, addressed using a glass-polymer hybrid technology in which poly(dimethylsiloxane), PDMS, is used as an elastomeric packaging over-layer. In particular, we describe the wide range of modelling and microfabrication processes required for the successful manufacture, integration and packaging of such arrays. The integration of both optical and fluidic circuits in this device avoids precise alignment requirements and results in a compact, robust and reliable device. Finally, in this paper, we describe the implementation of a pumping system for delivering small amounts of fluid across the array together with an optical signal treatment.     

The OpenPicoAmp: An Open-Source Planar Lipid Bilayer Amplifier for Hands-On Learning of Neuroscience

Understanding the electrical biophysical properties of the cell membrane can be difficult for neuroscience students as it relies solely on lectures of theoretical models without practical hands on experiments. To address this issue, we developed an open-source lipid bilayer amplifier, the OpenPicoAmp, which is appropriate for use in introductory courses in biophysics or neurosciences at the undergraduate level, dealing with the electrical properties of the cell membrane. The amplifier is designed using the common lithographic printed circuit board fabrication process and off-the-shelf electronic components. In addition, we propose a specific design for experimental chambers allowing the insertion of a commercially available polytetrafluoroethylene film. We provide a complete documentation allowing to build the amplifier and the experimental chamber. The students hand-out giving step-by step instructions to perform a recording is also included. Our experimental setup can be used in basic experiments in which students monitor the bilayer formation by capacitance measurement and record unitary currents produced by ionic channels like gramicidin A dimers. Used in combination with a low-cost data acquisition board this system provides a complete solution for hands-on lessons, therefore improving the effectiveness in teaching basic neurosciences or biophysics.     

Analyzing nested experimental designs—A user-friendly resampling method to determine experimental significance

While hierarchical experimental designs are near-ubiquitous in neuroscience and biomedical research, researchers often do not take the structure of their datasets into account while performing statistical hypothesis tests. Resampling-based methods are a flexible strategy for performing these analyses but are difficult due to the lack of open-source software to automate test construction and execution. To address this, we present Hierarch, a Python package to perform hypothesis tests and compute confidence intervals on hierarchical experimental designs. Using a combination of permutation resampling and bootstrap aggregation, Hierarch can be used to perform hypothesis tests that maintain nominal Type I error rates and generate confidence intervals that maintain the nominal coverage probability without making distributional assumptions about the dataset of interest. Hierarch makes use of the Numba JIT compiler to reduce p-value computation times to under one second for typical datasets in biomedical research. Hierarch also enables researchers to construct user-defined resampling plans that take advantage of Hierarch’s Numba-accelerated functions.     

Group screening for rare events based on incomplete block designs

Fields such as, diagnostic testing, biotherapeutics, drug development, and toxicology among others, center on the premise of searching through many specimens for a rare event. Scientists in the business of “searching for a needle in a haystack” may greatly benefit from the use of group screening design strategies. Group screening, where specimens are composited into pools with each pool being tested for the presence of the event, can be much more cost-efficient than testing each individual specimen. A number of group screening designs have been proposed in the literature. Incomplete block screening designs are described here and compared with other group screening designs. It is shown under certain conditions, that incomplete block screening designs can provide nearly a 90% cost saving compared to other group screening designs such as when prevalence is 0.001 and screening 3876 specimens with an ICB-sequential design vs. a Dorfman design. In other cases, previous group screening designs are shown to be most efficient. Overall, when prevalence is small (≤0.05) group screening designs are shown to be quite cost effective at screening a large number of specimens and in general there is no one design that is best in all situations. © 2018 American Institute of Chemical Engineers Biotechnol Progress, 35: e2770, 2019.     

A new efficient mixture screening design for optimization of media

Screening ingredients for the optimization of media is an important first step to reduce the many potential ingredients down to the vital few components. In this study, we propose a new method of screening for mixture experiments called the centroid screening design. Comparison of the proposed design with Plackett‐Burman, fractional factorial, simplex lattice design, and modified mixture design shows that the centroid screening design is the most efficient of all the designs in terms of the small number of experimental runs needed and for detecting high‐order interaction among ingredients. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Optimal DNA pooling-based two-stage designs in case-control association studies

Study cost remains the major limiting factor for genome-wide association studies due to the necessity of genotyping a large number of SNPs for a large number of subjects. Both DNA pooling strategies and two-stage designs have been proposed to reduce genotyping costs. In this study, we propose a cost-effective, two-stage approach with a DNA pooling strategy. During stage I, all markers are evaluated on a subset of individuals using DNA pooling. The most promising set of markers is then evaluated with individual genotyping for all individuals during stage II. The goal is to determine the optimal parameters (pi(p)(sample ), the proportion of samples used during stage I with DNA pooling; and pi(p)(marker ), the proportion of markers evaluated during stage II with individual genotyping) that minimize the cost of a two-stage DNA pooling design while maintaining a desired overall significance level and achieving a level of power similar to that of a one-stage individual genotyping design. We considered the effects of three factors on optimal two-stage DNA pooling designs. Our results suggest that, under most scenarios considered, the optimal two-stage DNA pooling design may be much more cost-effective than the optimal two-stage individual genotyping design, which use individual genotyping during both stages.     

Factorial and time course designs for cDNA microarray experiments

Microarrays are powerful tools for surveying the expression levels of many thousands of genes simultaneously. They belong to the new genomics technologies which have important applications in the biological, agricultural and pharmaceutical sciences. There are myriad sources of uncertainty in microarray experiments, and rigorous experimental design is essential for fully realizing the potential of these valuable resources. Two questions frequently asked by biologists on the brink of conducting cDNA or two-colour, spotted microarray experiments are 'Which mRNA samples should be competitively hybridized together on the same slide?' and 'How many times should each slide be replicated?' Early experience has shown that whilst the field of classical experimental design has much to offer this emerging multi-disciplinary area, new approaches which accommodate features specific to the microarray context are needed. In this paper, we propose optimal designs for factorial and time course experiments, which are special designs arising quite frequently in microarray experimentation. Our criterion for optimality is statistical efficiency based on a new notion of admissible designs; our approach enables efficient designs to be selected subject to the information available on the effects of most interest to biologists, the number of arrays available for the experiment, and other resource or practical constraints, including limitations on the amount of mRNA probe. We show that our designs are superior to both the popular reference designs, which are highly inefficient, and to designs incorporating all possible direct pairwise comparisons. Moreover, our proposed designs represent a substantial practical improvement over classical experimental designs which work in terms of standard interactions and main effects. The latter do not provide a basis for meaningful inference on the effects of most interest to biologists, nor make the most efficient use of valuable and limited resources.     

Ontogeny of Vision in Marine Crustaceans

Marine crustaceans present an extremely interesting set of examplesin which to examine visual development and metamorphosis. Larvaeof these animals are almost always planktonic, living in thelight field of open waters. The presence of a simple, predictablephotic environment, the relatively basic visual requirementsof larvae, and the need to remain transparent to reduce predationlead to the use of a single eye type throughout all marine crustaceanlarvae. Adult crustaceans, on the other hand, use a greaterdiversity of optical designs than all other animals combined,occupy habitats from the deep sea to mountaintops, and havevery complex visual systems and behaviors. Thus, visual developmentvaries tremendously among modern Crustacea. In this brief review,we consider the structure and development of marine crustaceaneyes, focusing on optics, retinal design, and metamorphosisof the visual pigments.     

A low‐cost,computer‐controlled robotic flower system for behavioral experiments

Human observations during behavioral studies are expensive, time‐consuming, and error prone. For this reason, automatization of experiments is highly desirable, as it reduces the risk of human errors and workload. The robotic system we developed is simple and cheap to build and handles feeding and data collection automatically. The system was built using mostly off‐the‐shelf components and has a novel feeding mechanism that uses servos to perform refill operations. We used the robotic system in two separate behavioral studies with bumblebees (Bombus terrestris): The system was used both for training of the bees and for the experimental data collection. The robotic system was reliable, with no flight in our studies failing due to a technical malfunction. The data recorded were easy to apply for further analysis. The software and the hardware design are open source. The development of cheap open‐source prototyping platforms during the recent years has opened up many possibilities in designing of experiments. Automatization not only reduces workload, but also potentially allows experimental designs never done before, such as dynamic experiments, where the system responds to, for example, learning of the animal. We present a complete system with hardware and software, and it can be used as such in various experiments requiring feeders and collection of visitation data. Use of the system is not limited to any particular experimental setup or even species.     

New construction for transversal design.

The study of gene functions requires the development of a DNA library of high quality through much of testing and screening. Pooling design is a mathematical tool to reduce the number of tests for DNA library screening. The transversal design is a special type of pooling design, which is good in implementation. In this paper, we present a new construction for transversal designs. We will also extend our construction to the error-tolerant case.     

Bioprocess monitoring

Electrochemical sensors for pH and dissolved oxygen remain the most commonly used in bioprocess monitoring, but continued research has resulted in improved optical sensors. Optical sensors for dissolved oxygen and dissolved carbon dioxide are now commercially available. Advances in optics and electronics are further driving down the costs of these sensors. In the near future, bioprocess optimization will change paradigms as massively parallel, fully instrumented bioreactors become available and high-throughput bioprocessing becomes a reality.     

Optimal design of genetic studies of gene expression with two-color microarrays in outbred crosses

Combining global gene-expression profiling and genetic analysis of natural allelic variation (genetical genomics) has great potential in dissecting the genetic pathways underlying complex phenotypes. Efficient use of microarrays is paramount in experimental design as the cost of conducting this type of study is high. For those organisms where recombinant inbred lines are available for mapping, the “distant pair design” maximizes the number of informative contrasts over all marker loci. Here, we describe an extension of this design, named the “optimal pair design,” for use with F₂ crosses between outbred lines. The performance of this design is investigated by simulation and compared to several other two-color microarray designs. We show that, for a given number of microarrays, the optimal pair design outperforms all other designs considered for detection of expression quantitative trait loci (eQTL) with additive effects by linkage analysis. We also discuss the suitability of this design for outbred crosses in organisms with large genomes and for detection of dominance.     

New constructions of one- and two-stage pooling designs.

The study of gene functions requires a DNA library of high quality, such a library is obtained from a large mount of testing and screening. Pooling design is a very helpful tool for reducing the number of tests for DNA library screening. In this paper, we present new one- and two-stage pooling designs, together with new probabilistic pooling designs. The approach in this paper works for both error-free and error-tolerance scenarios.