Design for Manufacturability (DFM) in the Life Sciences

Transcription

1 T E C H N I C A L N O T E Design for Manufacturability (DFM) in the Life Sciences Fluorescence Spectroscopy Product Platform Realized with TracePro TM Suite of Opto-Mechanical Design Software Tools Authors: Edward Freniere, Ph.D., Richard Hassler, Linda Smith from Lambda Research Corporation; Eric Heinz from Heinz Optical Engineering Company; Acknowledgement: Iain Johnson, Ph.D. from Invitrogen Corporation Optical Instrumentation Development for the Life Sciences Product Development The inherently interdisciplinary nature of developing instrumentation for the life sciences requires a high level of collaboration between scientists and engineers across the fields of analytical or clinical chemistry, optics, mechanics, material science and microbiology. Moreover, product development teams are competing for first-to-market benefits that are driven by intellectual property life times and insuring an installed base quickly to realize recurring consumable sales. Concurrently, they need to comply with current Good Manufacturing Practices (cgmp). Competitive advantage in the life sciences industry can be achieved by adhering to a design-for-manufacture (DFM) process where system level product performance specifications and design elements are communicated effectively and in compliance across the multiple technical disciplines. System level modeling enables adherence to the methodical design process without the cost and time associated with iterative hardware prototyping and laboratory and clinical testing. Fluorescent Probe Technologies Fluorescence probe, luminescent reporter, and nanocrystal technologies enable very sensitive detection of molecules and rapid detection of specific changes within target biological samples. The menu of fluorescence based biological assays is large and rapidly increasing with the burgeoning discovery of new biomarkers. The fundamental characteristics of this optical phenomenon, however, severely complicate instrumentation design. > Fluorescence Spectroscopy > Flow Cytometry > Micro Array Readers > Nucleic Acid Amplification > Assay & Cell Based Imaging > Confocal Laser Scanning Microscopy > Laser Induced Fluorescence Detection (LIF) > Fluorescence Resonance Energy Tranfer (FRET) > Biosensors > In-Vitro Diagnostics > In-Vivo Diagnostics Page 1

2 Fluorescence occurs when the molecular absorption of a photon triggers emission of a lower-energy photon with a longer wavelength. The observable Stokes shift is the difference between positions of the band maxima of the absorption and fluorescence emission spectra. A fluorophore is most efficiently excited by a specific wavelength. However, light of wavelengths near the excitation maximum also causes excitation but less efficiently. Fluorescence emission occurs at a specific wavelength. Excitation at higher or lower wavelengths affects only the intensity of the emitted light. Design Workflow Optical and mechanical assemblies are designed, analyzed and toleranced in commercially available lens design software and SolidWorks 3-D mechanical design software. Lens designs including parameterized geometry, tolerances and optical materials are translated with TracePro LensWorks into SolidWorks with the TracePro Bridge add-in installed. The single SolidWorks file is opened by TracePro. Biological samples, fluorophores, light sources, detectors, coatings and filter data are then specified in TracePro. TracePro performs system level modeling, analysis and optimization of light distributions, stray light, throughput, flux absorbed by surfaces and bulk media, polarization effects, and fluorescence effects. Design modifications made in the TracePro model are then updated in the archived SolidWorks file. All product development data and documentation compliance is managed within SolidWorks. Page 2

3 Case Study Fluorescence Spectroscopy Platform A Design-for-Manufacture (DFM) process is executed for a fluorescence spectroscopy product platform. A multi-disciplinary design team eliminates the cost of quality using a formal design method, facilitated by Lambda Research Corporation s TracePro suite of 3-D opto-mechanical design tools and SolidWorks 3-D mechanical design software. Platform development presents rigorous design challenges - from identifying optical layout and fluorophore alternatives - to minimizing the exponential cumulative effect of component quality and quantity - to tolerancing component dimensions - to complying with GMP documentation and traceability requirements. The design is highly constrained in terms of cost and the requirement to achieve both the sensitivity and dynamic range to detect the presence of a breadth of fluorescencetagged proteins. Fluorophore Selection and Biological Sample Model Based on the target protein, a fluorescent dye, Alexa Fluor 488 Dye, is selected that has the proper reactive groups and accommodates assay conditions including photostability and ph. Optical properties of the dye including relative excitation and emission curves and the peak molar extinction coefficient are retrieved from the Invitrogen Molecular Probes Products database resident in TracePro and TracePro Bridge for SolidWorks. The dye is modeled in an aqueous solution with a ph>8. Quantum efficiency is determined by biochemists to be 0.92 as a free dye and 0.55 when conjugated to the target protein. The initial design tests the feasibility of the fluorophore concentration at 10 E -10 moles per liter. A range of concentrations is modeled to validate adherence to the dynamic range specification. Page 3

4 Excitation Source and Detector Selection and Model Based on the peak excitation wavelength of Alexa Fluor 488 Dye, an excitation source is selected that balances a wavelength closest to peak excitation, luminous flux, and cost. A blue LED, Kingbright Model WP7524PBC/J, with wavelength of 467 ± 22nm is selected. Its SolidWorks model and optical properties are downloaded from and imported into the TracePro model. Luminous flux and directionality of the LED s output are characterized in lab and imported into the TracePro model. Based on the peak emission wavelength of the Alexa Fluor 488 Dye, 517 nm, a detector is selected that balances cost and responsivity. A silicon photodiode is selected and its spectral responsivity curve is imported into the TracePro model. Fluorescence Filtering Fluorescence filter sets are essential in separating the fluorescence emission photons at the detector from the more intense excitation photons from the source. It is necessary to reduce the excitation light intensity while maximizing the number of fluorescence emission photons. High capture efficiency enables reductions in overall excitation light levels and thus, reductions in dye photobleaching and phototoxicity of the biological sample. Filter selection is most often a complex analysis of the spectral relationships of fluorophores, optical filters, excitation sources and detectors. Because off-the-shelf components offer several ways to minimize costs in product development, purchasing, manufacturing, quality and reliability within in the DFM process, off-the-shelf filters available from commercial suppliers such as Newport Corporation and Omega Optical, Inc. are considered and modeled. Optical and mechanical characteristics of these off-the-shelf filters are imported from TracePro s libraries and modeled in the system. Narrowband excitation and emission filters centered on the Alexa Fluor 488 dye s absorption and emission peaks are selected to minimize spectral overlap of the emission signal with the excitation signal and increase signal isolation. Filter selection is further complicated by fluorophores significantly different spectral properties in a particular application such as nucleic acid stains bound to RNA than those of fluorophores in aqueous solution. Complex and application specific driven characteristics of the fluorophores sample preparation and biological target are then modeled in combination with the selected filter pair by iterating and analyzing Quantum Efficiency and fluorophore concentration values directly in the TracePro and SolidWorks model. Simulation and Analysis The complete opto-mechanical system, including the fluorescence-tagged biological sample, is modeled and documented in SolidWorks with the TracePro Bridge add-in. The single, archived SolidWorks file is then opened with TracePro for optical simulation and analysis. Source rays propagate through the model with portions of the flux of each ray allocated to absorption, specular reflection and transmission, fluorescence, polarization and scattering. Page 4

5 From the simulation, contributions to sensitivity are analyzed system throughput at detector, flux absorbed by surfaces and bulk material, stray and scattered light from mechanical and optical surfaces. Contributions to dynamic range are analyzed by changing the concentration and quantum efficiency of the dye to simulate the breadth of addressable sample preparations. From the TracePro irradiance map, it is concluded that the sensitivity specification is not achieved. From the TracePro flux report, it is concluded that there is: > Insufficient fluorescence emission photons captured > Unwanted stray light from optical and mechanicalcomponents reaching the detector Design for Manufacturability Component level improvements affecting system level sensitivity are identified relative to driving DFM factors such as component cost, number of components, and manufacturing tolerances. Page 5

6 From the TracePro and DFM analyses, it is concluded that the sensitivity specification may be achieved by increasing the f/# on both the LED and detector collection optics and modifying the mechanics to reduce stray light at the detector. These design modifications offer the largest improvement in performance at orders of magnitude less expense. The design of the new collection lenses is realized and toleranced in commercially available lens design software and translated via LensWorks into the single archived SolidWorks file. Mechanics are modified and a single set of documentation is updated in SolidWorks. The same SolidWorks file is then opened by TracePro for design validation. Summary The development of the fluorescence spectroscopy product platform demonstrates that with an integrated set of software design tools, a disciplined DFM product development process can be executed effectively and efficiently within a multidisciplinary design team. System level performance and component level specifications can be communicated across technical disciplines while data and design integrity can be insured by documenting in compliance with cgmp. Product cost and time to market are minimized with improved product quality and reliability. TracePro Bridge TM for SolidWorks Page 6 Lambda Research Corporation 25 Porter Road Littleton, MA USA Phone: Fax: sales@lambdares.com Lambda Research Corporation