WO2010052578A1 - Dispositifs et procédé visant à réduire l'autofluorescence de matières - Google Patents

Dispositifs et procédé visant à réduire l'autofluorescence de matières Download PDF

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Publication number
WO2010052578A1
WO2010052578A1 PCT/IB2009/007590 IB2009007590W WO2010052578A1 WO 2010052578 A1 WO2010052578 A1 WO 2010052578A1 IB 2009007590 W IB2009007590 W IB 2009007590W WO 2010052578 A1 WO2010052578 A1 WO 2010052578A1
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WO
WIPO (PCT)
Prior art keywords
source
autofluorescence
annealing
light source
filter
Prior art date
Application number
PCT/IB2009/007590
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English (en)
Inventor
Girgio Horak
Gian-Luca Lettieri
Martin O'keane
Piero Zucchelli
Original Assignee
Spinx, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Spinx, Inc. filed Critical Spinx, Inc.
Publication of WO2010052578A1 publication Critical patent/WO2010052578A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/12—Specific details about manufacturing devices

Definitions

  • a method of a treatment is used to eliminate material autofluorescence.
  • photobleaching or so-called autofluorescence annealing of the material is achieved by exposure to electromagnetic radiation in conjunction with suitable supplementary apparatus.
  • simultaneous heating or cooling along with the exposure to electromagnetic radiation may assist in the autofluorescence annealing process.
  • autofluorescence annealing photobleaching
  • process process
  • treatment treatment
  • UV or visible or IR is generated by a suitable apparatus, then additionally modified or reflected by optical filters, gratings or mirrors before irradiating and photobleaching a given work piece. It is contemplated within the scope of this disclosure that this process can also be achieved by irradiation with narrow-band electromagnetic radiation, as for example those generated by a monochromatic source or by a coherent source (lasers). This treatment according to the disclosure can be achieved using commercially available means, lends itself to an efficient stable production environment, and repeatable with a very high process yield.
  • FIG. 1 is a schematic of Process General Arrangement of Plastic Autoflourescence Annealing according to the disclosure
  • FIG. 2 is a schematic of multi-component device prior to final assembly according to the disclosure
  • FIG. 3. is a schematic of a multi-component device after final assembly according to the disclosure, the autofluorescence annealing process could be done on the fully assembled part, either with or without the protective packaging delivered to the end user;
  • FIG. 4. graphically shows the autofluorescence of certain materials
  • FIG. 5. shows the absorption spectrum of a polymeric material
  • FIG. 6. graphically depicts the autofluorescent emission spectrum from plastic
  • FIG. 7. shows the plastic transmittance of sheet material
  • FIG. 8. depicts the effect of sunlight exposure alone reducing the plastic autofluorescence by about 80-90%;
  • FIG. 9. schematically depicts how sunlight was compared to that of an UV source - in this case an iron lamp - in conjunction with a UVA filter irradiating the microfluidic device at a distance of 130mm;
  • FIG. 10. shows plastic autofluorescence with uv (365) excitation;
  • FIG. 11. depicts autofluorescence reduction
  • FIG. 12. depicts autofluorescence reduction by sun and with a UVA filter.
  • the present disclosure contains devices and methods for reducing the intrinsic autofluorescence of materials.
  • these materials may be transparent or translucent or opaque or reflective.
  • Appropriate materials for this process include, but are not limited to, inorganic and organic polymers (for example, Polymethyl methacrylate (PMMA) or Cyclo Olefin Polymer (COP), and or silicones such as Polydimethylsiloxane (PDMS), an organic silicon-based polymer) or glass or quartz (silicon dioxide, silica) and or composite materials (including metal matrix composites) and or metals (both ferrous and non-ferrous) and or ceramics and mixtures thereof.
  • PMMA Polymethyl methacrylate
  • COP Cyclo Olefin Polymer
  • silicones such as Polydimethylsiloxane (PDMS), an organic silicon-based polymer) or glass or quartz (silicon dioxide, silica) and or composite materials (including metal matrix composites) and or metals (both ferrous and non-ferrous) and or ceramics and mixtures thereof.
  • the autofluorescent annealing process according to the disclosure is done by exposure to electromagnetic radiation, meaning ultraviolet light or infrared light or visible light can be used. It is contemplated within the scope of the disclosure that this electromagnetic radiation may be broadband or narrowband in nature and may be generated by a single source or multiple sources. Heating or cooling of the material in conjunction with exposure to the aforementioned electromagnetic radiation may optionally be used during the process of autofluorescent annealing, but also as a preparation process or as a post-processing operation. The role of heat treatment could be understood in terms of generating or releasing mechanical stress on the material structure.
  • the electromagnetic radiation used in the process may be generated by man-made equipment or come from natural sources, such as sunlight.
  • the application of the autofluorescence annealed material may be to any device or receptacle or vessel or product used to hold a liquid or solid or gaseous sample which could be analyzed by optical detection methods, and in optical detectors themselves. Therefore, the application could include, but not be limited to, microfluidic devices, biological substrates, chemical substrates, microtitre plates, cuvettes, sample containers (including vials), test tubes, laboratory glassware, syringes, drug delivery systems, intravenous drip bags, receptacles used in diagnostic applications, receptacles used in bio-defense products, receptacles used for human vaccines and medication, receptacles used in medicine, microscopy or micro titer plate readers, medical imaging systems and scientific imaging systems.
  • optical detection methods used on the autofluorescence annealed material include, but are not limited to, fluorescence intensity or fluorescence resonance energy transfer (FRET) or luminescence or scintillation or Time Resolved FRET (TR-FRET) or fluorescence lifetime or fluorescence polarization.
  • FRET fluorescence intensity or fluorescence resonance energy transfer
  • TR-FRET Time Resolved FRET
  • the autofluorescence annealing process requires: a source of electromagnetic radiation; the material to be irradiated being placed at a suitable working distance from the source; ancillary equipment such as optical filters may be placed to between the source and the parts; and a selected exposure time "t", either in continuous or pulsed time structure.
  • EXAMPLE I Autofluorescent Annealing Of Plastics Microfluidic Devices
  • the fluorescent background is generated during the manufacturing of the plastic raw material, during the injection molding or during hot embossing where the plastic undergoes several stresses like high pressure, temperature variations or mechanical stresses.
  • the assembly of the plastic parts during the manufacturing of a functional microfluidic devices can also induce stress responsible for autofluorescence.
  • the autofluorescence is present in all wavelengths ranging from near UV to deep IR.
  • the autofluorescence becomes visible when an untreated plastic microfluidic device is irradiated at the wavelength used to excite a fluorescent tag in a given assay. Once excited, the tag typically emits a fluorescent signal at wavelength which is slightly shifted with respect to the excitation wavelength. However, the plastic microfluidic device also emits a fluorescent signal that increases the background noise at the wavelength of interest. This autofluorescent signal therefore reduces the signal to background ratio.
  • the autofluorescence of the plastic device can be reduced to zero or substantially close to zero for excitation wavelengths ranging from very low UV to the deep infra red wavelengths.
  • This autofluorescence annealing thus renders the plastic microfluidic device compatible with all commercial assays based on luminescence, HTRF, TR-FRET, fluorescence polarization and luminescence.
  • the elimination of plastic autofluorescence is thought to be due to photobleaching by irradiation of the plastic part with electromagnetic radiation, in this particular example using UV. Combined heating and UV irradiation may increase the efficiency of the photobleaching.
  • the method according to the disclosure is compatible with mass production of plastic parts for microfluidic applications. Most medical grade plastics have a high transmittance in the UV spectrum, and thus the process of photobleaching can be used to treat several microfluidic plastic parts simultaneously. Photobleaching can also be applied on the raw plastic material, to sub-components directly after injection molding or hot embossing or after the final device assembly process.
  • the autofluorescent annealing of plastic devices is taken as an example.
  • the process according to the disclosure requires: a UV light source, with one of a number of commercially available lamps being appropriate; the material to be irradiated being placed at a suitable working distance "d" from the UV source; an UVA filter between the UV source and material; and a selected exposure time "t".
  • the UVA filter is required when using a UV source to avoid an oxygen plasma reaction generated on the surface of the plastic microfluidic part facing the UV source.
  • the oxygen plasma generation could be functional to modifications of the polymer surface and could be even desirable in some applications.
  • Without a UVA filter the autofluorescence of the plastic decreases after one hour of exposure but does not disappear. Additional exposure, again without the UVA filter, provokes a new increase of the autofluorescence.
  • FIG. 1 A simplified process general arrangement according to the disclosure is depicted in FIG. 1.
  • the light source used for autofluorescence annealing of plastic parts according to the disclosure can be: a 400W iron lamp or a 1000 W iron lamp or a 2000W iron lamp or an iron lamp with a power below 400W or above 2000W; a 400W Gallium lamp or a IOOOW Gallium lamp or a 2000W Gallium lamp or a Gallium lamp with a power below 400W or above 2000W; a 400W mercury lamp or a IOOOW mercury lamp or a 2000W mercury lamp or a mercury lamp with a power below 400W or above 2000W. It is contemplated within the scope of this disclosure that other light sources known in the art can be used. Other sources that positively influence (reduce) the autofluorescence of the plastic include but are not limited to the following: sunlight, halogen lamps, neon lamps or indeed any source in the UV and visible range with sufficient power density.
  • Exposure time * at 400W t hours 1 3 - Number of parts * n - 1 6 -
  • the material to be autofluorescence annealed does not need to be arranged in a certain orientation, it is sufficient that they are exposed to the UV source at a suitable working distance.
  • the autofluorescent annealing process is typically carried out on fully assembled microfluidic devices, which are in a state in where the desired chemical, biochemical or biological assay could be run on said device.
  • the objects to be irradiated are placed at an appropriate working distance from the UV source.
  • the UVA filter is integrated into the UV source.
  • the autofluorescence annealing process according to the disclosure is simple to carry out, and does not require human supervision. According to the disclosure numerous general arrangements will achieve the desired level autofluorescence annealing of the parts to be irradiated. That is, sets of process parameters out with the recommended limits stated in the previous section could also produce the desired effect.
  • Permissible variations on the standard process include the following: the exposure time "t" can be reduced when using a higher power source of electromagnetic radiation, and vice versa with a less powerful source; modulation of the working distance between source and work piece(s) as a function of the source power; modulation of the heating power by varying the distance between source and work piece(s), and the spectrum of the source; simultaneous irradiation of multiple stacked parts and / or parts packed in a plastic bag e.g.
  • HDPE High Density Polyethylene
  • this plastic packaging could be of a protective nature around components from an upstream production step; conversely, the packaging could constitute the final packaging as delivered to the end user, protecting the fully assembled device up to the point where it is to be used; use of a diffuser in order to improve the efficiency of the treatment.
  • the product to be irradiated could be placed in a box whose walls are made of a diffusing material such as thin white paper; the parts could be placed in a suitable container (e.g. an open box) to aid diffusion, provided there is a clear path from the source of electromagnetic radiation to the parts, with the exception of course of the filters or gratings in between, the internal walls could be painted with a suitable paint to aid diffusion; in another illustrative embodiment, the products to be irradiated could be heated well above room temperature, in order to accelerate the autofluorescent annealing process, this heating would be done in tandem with exposure to electromagnetic radiation with appropriate ancillary equipment; in another embodiment, the products to be irradiated could be cooled to well below room temperature, to ensure there would be no danger of local material degradation due to heating from the source of electromagnetic radiation, this cooling would be done in tandem with exposure to electromagnetic radiation with appropriate ancillary equipment; in yet another embodiment, the filter or gratings could be omitted between
  • the autofluorescent annealing process may increase the hydrophilicity of the autofluorescence annealed material, and consequently change its' wettability. For microfluidic devices this could, for example, affect the shape of fluidic menisci inside the microfluidic structure. It is conceivable that this could, for instance, change the types and / or concentrations of buffers and / or detergents needed to run given assays.
  • the autofluorescent annealing process may increase the hydrophobicity of the autofluorescence annealed material, and consequently decrease its' wettability.
  • microfluidic devices this could, this could, for example, affect the fiction between fluid and microfluidic device, thereby change the minimum volume that could be reliably and repeatably dosed within the microfluidic device (dispensed, transferred from one area of the microfluidic structure to another).
  • Multiple sources of electromagnetic radiation may be used to increase process capacity or to irradiate opposing sides of a part. Parts may be transported past static source(s) of electromagnetic radiation by automated means.
  • the presence of a material(s) loaded with photo-absorbent material(s) whose absorption characteristics are relatively constant across the spectrum may negatively affect the process autofluorescence annealing.
  • the part could be irradiated by two or more sources of electromagnetic radiation, ensuring there is homogeneous autofluorescent annealing despite the presence of the aforementioned photo-absorbent material(s).
  • the presence of such material(s) loaded with a photoabsorbant material(s) whose absorption characteristics are relatively constant across the spectrum could be circumvented by appropriate choice of power of electromagnetic radiation source, working distance, number of sources, optical filter or grating type and exposure time.
  • the autofluorescent annealing process would reach a successful conclusion without having to explicitly take into account the presence of the aforementioned photo-absorbent material(s).
  • the constituent parts of the device could separately undergo the process of autofluorescent annealing prior to final assembly. That is, all constituent parts would be autofluorescence annealed individually before the final assembly was done.
  • FIG. 2 a schematic of multi-component device 201 prior to final assembly is depicted.
  • multi-component device 201 a top plate 203 and a bottom plate 205 are assembled with a plastic film 207 between the top plate 203 and bottom plate 205.
  • the autofluorescence annealing process according to the disclosure could be done on the constituent parts prior to final assembly.
  • FIG. 3 a multi-component device 301 after final assembly according to the disclosure is depicted. It is contemplated within the scope of the disclosure that the autofluorescence annealing process could be done on the fully assembled part, either with or without the protective packaging delivered to the end user.
  • an aluminum coated mirror - or indeed any mirror coating suitable for the reflection of the electromagnetic radiation utilized - would permit two or more sides of the part to be irradiated using one single source. When scaling the number of sources for larger production volumes, this could lead to a rationalization in the number of sources needed. 10
  • FIG. 5 shows the absorption spectrum of a plastic suitable for microfluidic devices.
  • the choice of material here is secondary, but the results illustrate an important general point: a given material can be all but transparent to a specific wavelength of incident light, yet absorb significant amounts of energy from another wavelength.
  • FIG. 5 shows the large absorption at around 20 325nm and low absorption at around 415nm.
  • the emission spectrum from plastic was measured during excitation with a 337nm source as depicted in FIG. 6. While there is a strong signal from the excitation source, there is also a significant emission from the plastic at other wavelengths in the visible spectrum, for example between 400 and 440nm, which could 25 compromise high sensitivity optical measurements from chemical, biological or biochemical assays in this range.
  • the excitation peak on the left of the histogram is due to Rayleigh scattering of the sample and the strength depends on which kind of solvent is used to prepare the sample (in this case the plastic was immersed in common biochemical buffer).
  • 337 nm was used as the excitation source because it is the wavelength of interest for many biological applications, for example in assays using Coumarins or TR-FRET detection.
  • FIG. 7 shows how much incident light is transmitted by the plastic, and therefore indirectly indicates how much energy is absorbed by it.
  • the plastic used was one suitable for 5 microfluidic devices using optical detection methods.
  • the plastic device tested was made up of two injection-molded plastic pieces.
  • the autofluorescence annealing process could equally be applied to other designs of microfluidic devices.
  • the autofluorescence annealing process has been applied to microfluidic devices both before and after final assembly.
  • the “reduction factor” is defined as the ratio of the fluorescent signal produced by the samples before exposure to radiation and the fluorescent signal produced by the sample after exposure to given irradiation. This factor being IX or 100%, it means that no reduction of the autofluorescence is achieved. This factor being larger than IX or 100%, for example 3X or
  • the measurement of the autofluorescence of the plastic after treatment was done using the test as shown in FIG 9 with "Violet laser” signifying a monochromatic source of 365nm light (bandwidth IOnm, FWHM (Full Width Half Minimum)),"half card” signifying an
  • the combination of iron lamp with UVA filter was much more efficient at photobleaching the plastic.
  • the results also indicate that the autofluorescent annealing effect indeed suppresses autofluorescent emissions across the spectrum from very low UV to deep IR.
  • UV irradiation in conjunction with a UVA filter reduces the autofluorescence of the plastic to zero or close to zero from very low UV to deep IR.
  • the reference used is the magnitude of autofluorescent emission from an identical microfluidic device that had not undergone the autofluorescent annealing process.
  • the lamp used in the following plots is an iron lamp of 400W power of the type previously described.
  • the first three bars refer to a UV and visible light source without a UVA filter.
  • the remaining two bars are varying exposure times to sunlight.
  • the long exposure to sunlight confirms that a lower irradiance - prolonged for a long time - has qualitatively similar effects with respect to a higher irradiance obtained with the lamp and the UVA filter but for a shorter time.
  • the process therefore can be optimized by suitable choice of spectral irradiance and exposure time.
  • the process according to the disclosure has advantages for users of microfluidic platforms or other platforms using detection, in that it allows chemical, biological, and biochemical processes to be run with lower volumes and /or concentrations than before, as the detection limit, signal to background and signal to noise ratios are reduced.
  • this treatment lends its self to application in a production environment for reasons such as: this treatment is cost effective, in that it uses commercially available light source, that is UV bulbs or similar; the treatment does not require human supervision; the treatment does not require very accurate process monitoring, for instance, if the exposure time is exceeded by 10% of the prescribed duration there is no negative effect on the parts; the treatment is a repeatable process with a high production yield; the treatment can be easily scaled to handle larger production volumes; the treatment lends itself to process automation, with plastic parts being irradiated by a static source whilst on an automatic conveyor system; it is possible to determine whether a plastic part has undergone the treatment or not by simply testing the autofluorescence; the treatment is a stable, irreversible process; and the process can be used to equalize the differences in optical properties of work pieces, ensuring good part homogeneity (both intra-batch and between different batches).

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un procédé de traitement destiné à supprimer l'autofluorescence d'une matière. Dans un aspect de l'invention, un blanchissage optique ou un "recuit d'autofluorescence" de la matière est mis en oeuvre par l'exposition de celle-ci à un rayonnement électromagnétique, conjointement avec un dispositif supplémentaire approprié. Le procédé de recuit d'autofluorescence peut comporter une opération de chauffage ou de refroidissement, mise en œuvre simultanément à l'exposition au rayonnement électromagnétique.
PCT/IB2009/007590 2008-11-06 2009-11-06 Dispositifs et procédé visant à réduire l'autofluorescence de matières WO2010052578A1 (fr)

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US61/111,928 2008-11-06

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018185672A1 (fr) * 2017-04-06 2018-10-11 Magbiosense Inc. Surfaces de capture d'essai biologique à autofluorescence décolorée
WO2023059483A1 (fr) * 2021-10-07 2023-04-13 Applied Materials, Inc. Procédés et systèmes de réduction d'auto-fluorescence dans des échantillons fluorescents

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007021809A2 (fr) * 2005-08-11 2007-02-22 Eksigent Technologies, Llc Systeme, dispositifs et procedes microfluidiques permettant de reduire l'autofluorescence d'arriere-plan et ses effets

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007021809A2 (fr) * 2005-08-11 2007-02-22 Eksigent Technologies, Llc Systeme, dispositifs et procedes microfluidiques permettant de reduire l'autofluorescence d'arriere-plan et ses effets

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018185672A1 (fr) * 2017-04-06 2018-10-11 Magbiosense Inc. Surfaces de capture d'essai biologique à autofluorescence décolorée
WO2023059483A1 (fr) * 2021-10-07 2023-04-13 Applied Materials, Inc. Procédés et systèmes de réduction d'auto-fluorescence dans des échantillons fluorescents

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