Increasing Productivity Through a Combination of Automation and Robotics: A Case Study of Assay Services

The Assay Services Department at Genentech, Inc., is a service laboratory that performs drug level measurement and antibody testing in support of pre-clinical animal studies and human clinical studies. As the number of Genentech products has increased, so have the number of studies, resulting in an annual increase in the number of samples generated and an increased demand for assay support. Assay Services has addressed this by increasing the efficiency and productivity of sample handling and assaying through the automation of various procedures. All sample dilutions are now done by automated dilutors, reducing the number of reassays and virtually eliminating the repetitive stress of manual sample dilutions. In addition, two complete ELISA robot stations have been in use over the last two years that have increased throughput by increasing the number of plates per run (a robot can assay ten to fifteen 96-well microtiter plates per run), and by allowing assays to run overnight without requiring the presence of lab personnel. The net gain from the automation efforts has been to double the number of samples run without increasing the number of lab personnel.     

Manufacturing prototypes for paper-based diagnostic devices

Diagnostic assays created using simple and inexpensive materials have the potential to transform healthcare management systems in resource-limited settings. This change can be caused by making assays more accessible (e.g., by improving portability) or by inspiring the development of tests that do not exist currently but would solve a considerable need. Microfluidic devices produced from patterned paper have emerged as a promising candidate for a general platform capable of supporting the development of these diagnostic assays. The ability to manufacture paper-based diagnostic assays reproducibly and in large volumes—potentially hundreds of millions of devices per year per assay—is a critical component of the successful translation of these tools out of the laboratory and into the hands of users. This subject, however, has been largely overlooked in lieu of efforts to develop new tools and techniques. In this review, we summarize the recent advances in two processes that are required to fabricate paper-based diagnostic assays: patterning microfluidic channels into paper and assembling devices. In addition, we discuss the advantages and disadvantages of each process in the context of scalability and potential use as a method to manufacture point-of-care devices.     

Automation of cell-based drug absorption assays in 96-well format using permeable support systems

Cell-based drug absorption assays, such as Caco-2 and MDCK-MDR1, are an essential component of lead compound ADME/Tox testing. The permeability and transport data they provide can determine whether a compound continues in the drug discovery process. Current methods typically incorporate 24-well microplates and are performed manually. Yet the need to generate absorption data earlier in the drug discovery process, on an increasing number of compounds, is driving the use of higher density plates. A simple, more efficient process that incorporates 96-well permeable supports and proper instrumentation in an automated process provides more reproducible data compared to manual methods. Here we demonstrate the ability to perform drug permeability and transport assays using Caco-2 or MDCKII-MDR1 cells. The assay procedure was automated in a 96-well format, including cell seeding, media and buffer exchanges, compound dispense, and sample removal using simple robotic instrumentation. Cell monolayer integrity was confirmed via transepithelial electrical resistance and Lucifer yellow measurements. Proper cell function was validated by analyzing apical-to-basolateral and basolateral-to-apical movement of rhodamine 123, a known P-glycoprotein substrate. Apparent permeability and efflux data demonstrate how the automated procedure provides a less variable method than manual processing, and delivers a more accurate assessment of a compound's absorption characteristics.     

SOS–A sample ordering system for delivering “assay-ready” compound plates for drug screening

Many bottlenecks in drug discovery have been addressed with the advent of new assay and instrument technologies. However, storing and processing chemical compounds for screening remains a challenge for many drug discovery laboratories. Although automated storage and retrieval systems are commercially available for medium to large collections of chemical samples, these samples are usually stored at a central site and are not readily accessible to satellite research labs.Drug discovery relies on the rapid testing of new chemical compounds in relevant biological assays. Therefore, newly synthesized compounds must be readily available in various formats to biologists performing screening assays. Until recently, our compounds were distributed in screw cap vials to assayists who would then manually transfer and dilute each sample in an “assay-ready” compound plate for screening. The vials would then be managed by the individuals in an ad hoc manner.To relieve the assayist from searching for compounds and preparing their own assay-ready compound plates, a newly customized compound storage system with an ordering software application was implemented at our research facility that eliminates these bottlenecks. The system stores and retrieves compounds in 1 mL-mini-tubes or microtiter plates, facilitates compound searching by identifier or structure, orders compounds at varying concentrations in specified wells on 96- or 384-well plates, requests the addition of controls (vehicle or reference compounds), etc. The orders are automatically processed and delivered to the assayist the following day for screening. An overview of our system will demonstrate that we minimize compound waste and ensure compound integrity and availability.     

Identify predictors of university students’ continuance intention to use online carbon footprint calculator

An online carbon footprint calculator (CFC) is a Green Information System, which enables individuals to self-estimate carbon footprints, self-manage behaviour, and accordingly self-control carbon emissions. Individuals’ persistent CFC use may help to slow down global warming. Thus, it is vital to establish a model to investigate the motivational predictors of CFC continuance intention. We formulated a model by integrating Expectation-Confirmation Model (ECM) and Theory of Planned Behaviour (TPB), with the former measuring continuance intention and the latter measuring low-carbon behavioural intention. To validate the model and identify significant predictors, a survey was conducted on undergraduate students. The survey results revealed that ECM accounted for 50% of the variance in continuance intention, that TPB accounted for 35% in low-carbon behavioural intention, and that the integrated model accounted for 64% in CFC continuance intention. Plus, direct and significant predictors of CFC continuance intention included low-carbon attitude, low-carbon behavioural intention, low-carbon subjective norm, satisfaction, and usefulness. Findings, implications, and suggestions are accordingly offered.     

GigaMatrix: an ultra high-throughput tool for accessing biodiversity

Recombinant approaches for tapping into the biodiversity present in nature for the discovery of novel enzymes and biosynthetic pathways can result in large gene libraries. Likewise, laboratory evolution techniques can result in large but potentially valuable libraries. Thorough screening of these libraries requires ultra high-throughput methods. The GigaMatrix^™ screening platform addresses this opportunity using reusable high-density plates with 100,000 to 1,000,000 through-hole wells in a microplate footprint. In addition to throughputs of over 10⁷ wells per day, the platform offers a significant reduction in reagent use and waste, has fully integrated automated “cherry picking,” and uses no complicated dispensing equipment. Wells containing putative hits from targeted fluorescent liquid phase assays are revealed by a fluorescent imaging system. Vision-guided robotics are utilized to recover hits by accessing individual 200 μm and smaller wells with a disposable sterile needle. The GigaMatrix platform has proven to be an effective and efficient tool for screening gene libraries for both discovery and evolution applications.     

Corning® 384 Well Low Volume Microplate Performance in Miniaturized Assays

In response to the ever-increasing demand for microplates that facilitate assay miniaturization without compromising assay integrity, Corning's new 384 well Low Volume (384 LV) microplates were evaluated using a homogenous, fluorescence polarization protease assay. Fluorescently labeled casein substrate was incubated over time with dilutions of protease at working volumes that range from 1 to 20 μL and reagent concentrations that range from 0.125 to 50 pg/μL. Four types of plates were evaluated:

• Corning 384 well LV black medium bind

• Corning 384 well LV black non-binding surface (NBS™)

• Corning 1536 well black medium bind

• Competitor 384 well small volume

Protease activity was detected as a decrease in polarized fluorescence resulting from substrate degradation over time. Following assay miniaturization to 20 μL working volumes and protease concentration reduction from 25 to 0.25 pg/μL, Corning 384 well LV medium bind and NBS coated assay plates maintained superior signal to noise ratios compared to competitor's 384 well small volume assay plates. At even lower 10 μL working volumes, Corning 384 well LV medium bind assay plates maintained signal to noise ratios comparable to the competitor's assay plates. However as the reagent concentrations were further reduced to 2.5 pg/μL, Corning 384 well LV medium bind and NBS coated assay plates had significantly greater signal to noise ratios compared to the competitor's assay plates. As working volumes were reduced even further to 5 μL and 1 μL, Corning 384 well LV medium bind and NBS coated assay plates had significantly greater signal to noise ratios than the competitor's 384 well small volume assay plates. Furthermore, at 1 μL working volumes, Corning 384 well LV NBS coated assay plates had significantly greater signal to noise ratios than the competitor's assay plates and standard 1536 well assay plates.These results suggest that Corning 384 well LV medium bind and NBS coated assay plates facilitate assay miniaturization while maintaining assay integrity. Furthermore, as working volumes were significantly reduced to those typically found in 1536 deep well and standard 1536 well assay plates (5 and 1 μL, respectively), Corning's 384 well LV NBS coated assay plates provided superior assay performance.     

Performance evaluation of 3D polystyrene 96-well plates with human neural stem cells in a calcium assay

In this study, we have generated a high-throughput screening (HTS)-compatible 3D cell culture platform by chemically "welding" polystyrene scaffolds into standard 2D polystyrene 96-well plates. The variability of scaffolds was minimized by introducing automation into the fabrication process. The fabricated 3D cell culture plates were compared with several commercially available 3D cell culture platforms with light and scanning electron microscopy. Voltage-gated calcium channel functionality was used to access the Z' factors of all plates, including a 2D standard plate control. It was found that with the No-Wash Fluo-4 calcium assay and neural progenitor cells, all plates display acceptable Z' factors for use in HTS. The plates with "welded" polystyrene scaffolds have several advantages, such as being versatile and economical, and are ready to use off the shelf. These characteristics are especially desired in HTS preclinical drug discovery applications.     

Supervised term weighting centroid-based classifiers for text categorization

In this paper, we study the theoretical properties of the class feature centroid (CFC) classifier by considering the rate of change of each prototype vector with respect to individual dimensions (terms). We show that CFC is inherently biased toward the larger (dominant majority) classes, which invariably leads to poor performance on class-imbalanced data. CFC also aggressively prune terms that appear across all classes, discarding some non-exclusive but useful terms. To overcome these CFC limitations while retaining its intrinsic and worthy design goals, we propose an improved centroid-based classifier that uses precise term-class distribution properties instead of presence or absence of terms in classes. Specifically, terms are weighted based on the Kullback–Leibler (KL) divergence measure between pairs of class-conditional term probabilities; we call this the CFC–KL centroid classifier. We then generalize CFC–KL to handle multi-class data by replacing the KL measure with the multi-class Jensen–Shannon (JS) divergence, called CFC–JS. Our proposed supervised term weighting schemes have been evaluated on 5 datasets; KL and JS weighted classifiers consistently outperformed baseline CFC and unweighted support vector machines (SVM). We also devise a word cloud visualization approach to highlight the important class-specific words picked out by our KL and JS term weighting schemes, which were otherwise obscured by unsupervised term weighting. The experimental and visualization results show that KL and JS term weighting not only notably improve centroid-based classifiers, but also benefit SVM classifiers as well.     

Functional cardiotoxicity profiling and screening using the xCELLigence RTCA Cardio System

Cardiac safety testing of lead drug candidates is an important part of the drug discovery and development process. All new chemical entities need to be subjected to extensive preclinical assessment for cardiac liability, especially for a potentially fatal form of ventricular arrhythmia referred to as Torsades de Pointes. We have developed an innovative label-free, real-time system, the xCELLigence RTCA Cardio System, which is designed to monitor contractility of cardiomyocytes based on impedance measurement. The assay is performed using specially designed microtiter plates that are integrated with gold microelectrodes. The system was validated using mouse embryonic stem cell-derived cardiomyocytes, human-induced pluripotent stem cell-derived cardiomyocytes, and rat neonatal primary cardiomyocytes by applying a variety of tool compounds and drugs with known mechanisms of action. Our data show that the time resolution in the assay can provide important information about compound action. Furthermore, the impedance-based beating profile in response to compound treatment can provide mechanistic toxicity information regarding the target being modulated and may be able to flag pro-arrhythmic compounds. We believe the real-time and kinetic aspect of this technology combined with beat-to-beat measurement of cardiomyocyte contraction would make this instrument an important part of preclinical cardiac safety assessment.     

The 104-well microplate

The 96-well microplate is a ubiquitous tool in the laboratory; its use is so extensive that in a limited number of situations it can be restrictive. Consider the situation where 96 samples need analysis or a downstream process in which the 96-well format leaves no space for additional standards or controls in the upstream 96-well processing. Consequently, plates are split or sample number reduced thereby incurring additional cost for plates, reagents, standards, controls, sample tracking, data files, and time to analyze the entire plate. A simple solution is proposed with the development of a companion 8 × 13-array microplate. The 104-well microplate was developed within the American National Standards Institute/Society for Biomolecular Science standards as to plate geometry and dimension, including well spacing (9 mm) with the exception that the columns have been shifted 4.5mm to the left to accommodate the 13th column. The extra column allows for additional standards/controls without modifying chemistry, incorporating additional plates or changing to a 384-well plate. We show negligible difference (-0.0003 optical density) when comparing mean absorbance readings in 96- and 104-well format. We demonstrate use of the 104-well plate in a 96-well environment by incorporating it in an enzyme-linked immunosorbent assay on a standard liquid handler. Results from the assay show no difference between formats (y=1.039x-0.004, r=0.997). Although the 104 plate was not created to supplant the 96-well standard, we conclude that the 104 plate can be incorporated into the 96-well environment without significant change in existing systems.     

人工神经网络方法预测乙烷氟氯衍生物的常压沸点

用ＢＰ算法，采取两个惯性项的惯性主以重系数方法和新的学习步长调整法，对乙烷氟氯衍生物的常压沸点作预测，根据预测出的沸点数据提指出了ＣＦ１１，ＣＦＣ１２和ＣＦＣ１１３的一些可能的代用品。由于采用新的训练策略，网络的收敛速度得到提高。     

Topological material layout in plates for vibration suppression and wave propagation control

We propose a topological material layout method to design elastic plates with optimized properties for vibration suppression and guided transport of vibration energy. The gradient-based optimization algorithm is based on a finite element model of the plate vibrations obtained using the Mindlin plate theory coupled with analytical sensitivity analysis using the adjoint method and an iterative design update procedure based on a mathematical programming tool. We demonstrate the capability of the method by designing bi-material plates that, when subjected to harmonic excitation, either effectively suppress the overall vibration level or alternatively transport energy in predefined paths in the plates, including the realization of a ring wave device. Most of this work was performed while AAL was employed at the Department of Mechanical Engineering.     

Automated High Throughput Screening of Fluorescent Imaging Plate Reader (FLIPR) Assays Using Assay Platform™ Integrated with Activity Base for ‘on the fly’ Compound Reconfirmation

Advances in the field of automation have meant hitherto complex manual cell-based assays can now be automated. These improvements have brought significant enhancements in throughput, data fidelity and consistency, and allowed a reallocation of constrained resources.Building upon these improvements, we have linked our automated cell-based screening system, Assay Platform™, to Activity Base (IDBS), a software package designed to automate the analysis of HTS data. Customisation of this package has resulted in software that can identify ‘active’ compounds and re-pick them ‘on the fly’ from the original compound plates for triplicate re-testing without operator intervention.Based on an operator initially defining ‘normal’ parameters for assay activity in Activity Base, combined with an automated quality control software module that checks data fidelity, wells containing ‘active’ compounds can be re-picked and re-tested at the end of an automated screening run. Automating cell-based assays has significantly improved productivity, and, with the synergism of Activity Base, has given us greater power to complete each screening run and report ‘active’ compounds to Chemistry more rapidly. This article presents our approach to the automation of cell-based Fluorescent Imaging Plate Reader (FLIPR) screening together with automated active re-test confirmation using Activity Base.     

A fuzzy neural network model and its hardware implementation

A fuzzy classifier based on a four-layered feedforward neural network model is proposed. This connectionist fuzzy classifier, called CFC, realizes the weighted-Euclidean-distance fuzzy classification concept in a massively parallel manner to recognize input patterns. CFC employs a hybrid supervised/unsupervised learning scheme to organize referenced pattern vectors. This scheme not only overcomes the major drawbacks of multilayer perceptron models using the backpropagation algorithm, i.e., the local minimal problem and long training time, but also avoids the disadvantage of the huge storage space requirement of the probabilistic neural network. According to experimental results, CFC shows better accuracy for speech recognition than several existing methods, even in a noisy environment. Moreover, it has higher stability of recognition rates for different environmental conditions. A massively parallel hardware architecture has been developed to implement CFC. A bus-oriented multiprocessor, systolic processor structure, and pipelining are used to obtain low-cost, high-performance fuzzy classification     

Construction of CFC-Programs by LTL-Specification

This article continues a cycle of papers, which describe an approach to construction and verification of discrete PLC-programs by an LTL-specification. The approach provides a possibility of PLC-program correctness analysis by the model checking method. For the specification of the program behaviour the linear-time temporal logic LTL is used. The correctness analysis of an LTL specification is performed automatically by the symbolic model checking tool Cadence SMV. It was previously shown how ST-, LD- and IL-programs are constructed by a correct (with verified program properties) LTL-specification. In this article, a technology of CFC-program construction by an LTL-specification is described. The language CFC (Continuous Function Chart) is a variation of FBD (Function Block Diagram). FBD is a graphical language for microcircuits. CFC provides a possibility of free allocation of program components and connections on a screen. The approach to construction of CFC-programs is shown by an example. PLC-program representation on CFC within the approach to programming by LTLspecification differs from other representations. It gives the visualization of a data flow from inputs to outputs. The influence and dependence among variables is explicitly shown during the program execution within one PLC working cycle. In fact, CFC-program is a scheme of PLC-program data flow.     

Boston Innovation''s SmartPlate Integrates Laboratory Functions to Increase Efficiency and Flexibility of Drug Discovery

Assay development is often facilitated by testing the chemistry against a representative or focused library of molecular compounds. Boston Innovation Inc. (“BII”), has introduced its SmartPlate technology which integrates molecular compound storage and dispensing in a single tool which enables many assay iterations quickly, easily, and at low cost, while maintaining consistent compound integrity. SmartPlate does this by storing concentrated compounds in 100% DMSO in sealed reservoirs, each with a dedicated dispensing tap. The compounds are never exposed to air, moisture, or light. Risks of cross-contamination and the need for cleaning or disposing pipette tips are eliminated. Compounds are metered and integrally diluted within the dispensing taps using BII's Direct Dilution™ technology, which allows SmartPlate to serve the needs of high density assay platforms with non-contact dispensing from 5 to 200nL of compound directly to standard assay plates. The integration of functions cuts waste, cost, and time from the drug discovery process by maintaining compound integrity, preserving valuable compound inventory, and eliminating process steps. At 15nL compound dispensing, 15μL of each compound in SmartPlate can now theoretically support one thousand assays without revisiting the main compound repository.     

The use of ‘Clickers’ in the classroom: Teaching innovation or merely an amusing novelty?

‘Clickers’ are individual response devices in which students each have a remote control that allows them to quickly and anonymously respond to questions presented in-class. Clickers are now being used in many classrooms as an active learning component of courses. Educators considering the use of clickers in their own classrooms may wonder whether the clickers are a worthwhile, pedagogical tool or merely an amusing novelty. As Li (2008) pointed out, research has examined clicker effects on interaction within the classroom, but little research has examined whether clicker use can affect the understanding of concepts. This article will discuss ways in which clickers may help students organize and understand material presented in the classroom. The paper is intended to help guide educators in potentially effective uses of clickers as well as to guide future research.     

Transfer of low nanoliter volumes between microplates using focused acoustics—automation considerations

Acoustic droplet ejection (ADE) gently and precisely aliquots nanoliter and picoliter liquid volumes without any physical contact with the solution being transferred. The technology is very automation-friendly, as it is compatible with conventional microplates. Focused energy from an acoustic transducer induces droplet ejection into an inverted standard microplate. The commercial system transfers low-nanoliter volumes of dimethyl sulfoxide–dissolved compound libraries and thereby enables cell-based assays to be performed in 1536-well plates.