|Publication number||USRE39772 E1|
|Application number||US 10/225,687|
|Publication date||Aug 14, 2007|
|Filing date||Mar 19, 1997|
|Priority date||Mar 19, 1996|
|Also published as||CA2248185A1, EP0928416A1, EP0928416A4, US6108463, WO1997035176A1|
|Publication number||10225687, 225687, PCT/1997/4398, PCT/US/1997/004398, PCT/US/1997/04398, PCT/US/97/004398, PCT/US/97/04398, PCT/US1997/004398, PCT/US1997/04398, PCT/US1997004398, PCT/US199704398, PCT/US97/004398, PCT/US97/04398, PCT/US97004398, PCT/US9704398, US RE39772 E1, US RE39772E1, US-E1-RE39772, USRE39772 E1, USRE39772E1|
|Inventors||James N. Herron, Douglas A. Christensen, Victor A. Pollack, Richard D. McEachern, Eric M. Simon|
|Original Assignee||University Of Utah Research Foundation|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (13), Non-Patent Citations (2), Referenced by (15), Classifications (21), Legal Events (2)|
|External Links: USPTO, USPTO Assignment, Espacenet|
This application is an application under 35 U.S.C. § 371 of PCT/US97/04398 filed on Mar. 19, 1997, claiming priority from U.S. Provisional patent application No. 60/022,434 filed on Aug. 8, 1996 and U.S. Provisional patent application No. 60/013,695 filed on Mar. 19, 1996.
This invention relates generally to components of a diagnostic apparatus, and more particularly to an improved biosensor having a lens (“waveguide”) and associated flow cell.
International Application No. PCT/US94/05567 (International Publication No. 94/27137, published Nov. 24, 1994) to the University of Utah Research Foundation discloses an apparatus for multi-analyte homogeneous, fluoroimmunoassays. In one embodiment, the application discloses an apparatus which uses a biosensor having a planar waveguide sandwiched, with an associated gasket, between two plates (
Unfortunately, the waveguide portion of integrally formed or molded biosensors may exhibit deformation upon fabrication, or warping during storage or temperature changes. Also, gaskets may not reliably seal or are not always sufficiently inert to reactants, and thus may interfere with the desired analysis.
It would be desirable to have a biosensor having reservoirs with inert walls, the walls being readily detachable from the waveguide so that one waveguide could be readily exchanged for another.
The invention includes a biosensor with a reservoir or reservoirs, the biosensors including a waveguide placed (e.g. “sandwiched”) between a plurality of members such as plates, at least one of the members being formed to define the walls of the reservoir or reservoirs where the reaction to be analyzed takes place. The reservoir walls are preferably an inert, opaque material such as a passivated metal (e.g., black anodized aluminum). Although the biosensor may include a gasket, the gasket is associated with the plurality of members and waveguide in such a way (e.g. by recessing the gasket into a channel formed into a metal plate) so that the gasket does not form any significant portion of the reservoir wall. Waveguides of varying composition (e.g. plastic, quartz, glass or siliconoxynitride) may be associated with the members to form the biosensor. A lens or lenses may be integrated with the waveguide. The metal plate of the biosensor has input and output ports for infusing, draining, or oscillating the liquid to be analyzed in the reaction reservoir.
Due to the sandwiching of the waveguide in between the members, the planar waveguide is generally less distorted than that of an integrally formed biosensor. A reaction to be analyzed is not interfered with due to the use of opaque, inert metal to structurally define the reservoir.
The biosensor design is advantageously configured to interact with a flat waveguide having a rear integrated lens design for reading light passing through the waveguide (not fluorescent/evanescent light, but reading the core laser beam light) to monitor coupling efficiency and beam quality. The invention thus also includes a flat waveguide associated with a rear lens to couple light out of the waveguide (and a biosensor using such a lens) to serve as a quality control measure, thus insuring that the biosensor is properly placed and that the light source is working.
The invention also includes orienting the biosensor in a particular position relative to an optical reading device and laser which increases the performance of the biosensor to the point where, surprisingly, whole blood can be quickly analyzed.
In the drawings, which depict presently preferred embodiments of the invention and in which like reference numerals refer to like parts in different views:
A. Flow Cell
The flow cell top, generally 100, depicted in
A design with at least two individual reservoirs has significant advantages over a single reservoir embodiment for instances when it is desirable to measure the test sample fluorescent simultaneously with fluorescence from a “control” region on the same waveguide. For example, the level of non-specific binding to the waveguide (or non-specific fluorescence) can be subtracted from test sample fluorescence. Also, measurement changes due to fluctuations in intensity of the exciting light can be corrected. In a displacement assay, the “control” region could be a pre-loaded waveguide with no analyte present in the sample, or with a known amount of analyte. With the depicted embodiment of three or more wells, fluorescence can be measured for both a no-analyte control and at least one known calibration analyte sample in addition to the “unknown” or test sample. Although, even with a single reservoir, the invention is able to analyze multiple analytes in a single sample (e.g. by use of a single waveguide in multiple experiments).
In the depicted embodiment, the reservoirs 102-106 have respective inlet/outlet apertures 108, 110, 112, 114, 116, 118 extending through the flow cell top 100 for injecting and withdrawing the liquid to be analyzed into the reservoirs 102-106. In some cases, this liquid may be oscillated into and out of the reservoir with a pump, which enhances the mixing of the analyte and reactant (see, e.g., FIG. 23). With oscillation, the performance (e.g. speed) of the assay is increased. In the depicted embodiment, each port 108-118 is associated with its own depressed recess formed 120, 122, 124, 126, 128, 130 in the flow cell top 100.
Between the recesses associated with a particular reservoir, lateral or longitudinal channels may be formed in the flow cell top to aid in mixing the liquid contained within the reservoir (not shown).
The outer periphery of the reservoirs 102-106 are each defined by respective walls 132, 134, 136 which are preferably integrally formed with the rest of the flow cell top 100, although they may be a separate component of the flow cell top. The inner circumferences 138, 140, 142 of the walls 132-136 are made of an inert, opaque material such as an inert, opaque plastic, or a metal such as passivated, black anodized aluminum, copper, stainless steel, or similar alloy. In the depicted embodiment, the entire flow cell top 100 is made of a metal, while in other embodiments (not shown), the flow cell may be made of a non-metallic material, and an opaque, dark material or metal sleeve placed within the reservoirs (not shown). Material in contact with the liquid should exhibit low protein absorption properties. Accordingly, a metal, a hydrophilic non-metallic material or a hydrophobic non-metallic material coated with a thin film of hydrophilic material (e.g. PEG, PLURONICS or other hydrogels) may be used.
In the depicted embodiment, the apertures 108-118 associated with the respective reservoirs 102-106 fluidically communicate the recessed portions 120-130 of the reservoirs with a pair of respective receptacles 144, 146, 148, 150, 152, 154 (shown by construction lines in
As depicted in FG. 4, the fluid inlet/outlet ports 156, 158 may be male threaded nipples which interact with corresponding threaded members (threads not shown) bored into the flow cell top 100. The open ends of the nipples are in fluid communication (e.g. by tubing or other conduit—
As further depicted in
B. The Waveguide
As depicted in
The edge of the planar portion has a receiving region (e.g. lens 166) for receiving light to be integrally propagated. In the embodiment depicted in
In another embodiment (not shown), the lens (or lenses) is not integrally associated with the waveguide, but is adapted to interact optically with the waveguide, or multiple waveguides.
Alternatively, rather than using a lens to coupling light into the waveguide, a grating could be used. Various gratings as well as methods for incorporating them into a waveguide are known. See, e.g., U.S. Pat. No. 5,480,687 (Jan. 2, 1996) to Heming et al. at column 4, lines 1-10, and column 6, line 20 to column 7, line 55, U.S. Pat. No. 5,081,012 (Jan. 14, 1992) to Flanagan et al., U.S. Pat. No. 5,455,178 (Oct. 3, 1995) to Fattinger, U.S. Pat. No. 5,442,169 (Aug. 15, 1995) to Kunz, and U.S. Pat. No. 5,082,629 (Jan. 21, 1992) to Burgess, Jr. et al. Gratings may be fabricated by a number of means including but not limited to: embossing, molding, photolithography, direct etch electron beam lithography, interference lithography, and phase shift lithography. Embossed gratings are mechanically stamped or thermally imbued onto a surface and thereupon affixed to a substrate. Photolithographic gratings are formed from the chemical development and etching of photoresist and substrate after masked illumination by an appropriate source. Interference and phase shift lithography are similar techniques which allow finer resolution of etched structures than does conventional photolithography. Ion or particle beam methods fabricate precise gratings by directly etching or “writing” a grating substrate with a stream of ions or molecular particles.
The grating itself can consist of an etched pattern of regular features in a metal film coated onto the planar portion of the waveguide or the front ramp. Standard diffraction gratings such as those used in spectrometers like “replica” gratings (gratings comprised of a dried epoxy coated with metal) can be used. The use of such grating couplers helps to avoid fabrication complexities associated with the use of a receiving lens or plasma-etched gratings. The procedure for applying such couplers is presently used to emboss holograms onto plastic credit cards, and, using such a process, the coupler could be mass produced at a relatively low cost.
In an alternative embodiment shown in
From efficiency measurements, it can be determined that for an integrated optical waveguide-fluoroimmunoassay, the most efficient etch depths are about 1.5 times that of the grating period. For diffraction to occur in a grating, the period d should be on the order of the wavelength of light (lambda). Given the pathlength difference, δ, between the light rays from two neighboring grating features (slits, rigids, and the like), a constructive interference pattern is established by the light leaving the grating when δ is an integer multiple, m, of the wavelength.
wherein d is the grating period, θt and θi are the transmitted and incident angles at the grating interface (measured relative to the surface normal), and nt and ni are the refractive indices of the transmitting and incident mediums (i.e. the waveguide and the substrate). Using this formula, one determines that the incident angle for coupling 632.8 nm light is 38.03° when the grating period is 0.7 μm.
The angle of incidence of light from air into the lowest order made of the waveguide and the groove density into waveguide films can be calculated by the use of the equation above, and was determined to be 4.6°, 27.4° and 57.2° for polystyrenes having densities of 2400 g/mm, 1800 g/mm, and 1200 g/mm, respectively, for incident light of 632.8 μm wavelength.
In still other embodiments, laser light may be prism-coupled onto an integrated optic waveguide (“IOW”) (not shown), end-fire coupled (i.e. direct focusing of light into the waveguide), or taper-coupled (e.g. by use of an adlayer film tapered in thickness or refractive index, preferably in conjunction with a grating coupler) into the waveguide (also not shown).
In order to taper-couple light into the flow cell, a gentle tapered section (e.g. either curved or linear) can be used to “funnel” light into the end of a thin planar waveguide. A well-collimated input beam (e.g. about 50 μm in thickness) due to the “Law of Brightness” constraint (i.e. the product of the beam extent and numerical aperture is a constant through the taper). The taper may be also coupled with a lens.
The waveguide depicted in
Laser light preferably enters the receiving lens 166 at mean angle θ (FIG. 8). The mean angle θ will typically vary dependent upon the type of material used 5 to form the waveguide and the optical properties of the media opposite both faces of the waveguide. When the waveguide or waveguide layer is made of polystyrene (e.g. NOVOCOR), then the mean angle will generally be less than 32°, e.g. 15° to 25°. Typical beam widths vary from 0.5 to 2.0 mm.
On the other side of the waveguide, an outcoupling 188 interacts with the rear or output lens 168 to ensure that light is detected (FIG. 8). The outcoupling 188 may be a single photodetector, multiple photodetectors, standard CCD (charge-coupled device) or like device. The light passing through the waveguide 164 and received by the outcoupling 188 is analyzed for quality and/or intensity. Unlike the end collection of light described in U.S. Pat. No. 4,582,809 to Block et al. (Apr. 15, 1986), in the present invention, the light may be detected at the end of the waveguide for two reasons. The first reason is as a quality control measure. The light passing through the waveguide may be measured so that the operator of the device knows that the biosensor has been properly placed in the apparatus and that the light source is still working. Alternatively, the device may be configured so that a predetermined strength of light must first be detected at the rear lens 168 before the apparatus will operate, again to ensure that the flow cell assembly (“biosensor”), generally 190, has been properly placed. The second reason for end detection involves calibration of the device to ensure that the amount of light travelling through the waveguide is uniform and, if it is not uniform to accommodate any differences. The light outcoupled from the lens 168 associated with the rear of the waveguide is preferably measured over the width of the lens to ensure that sufficient light is passing through the lens to create detectable fluorescence.
Preferably, a plastic waveguide such as that depicted in
Although the front lens ramp 192 and rear lens ramp 194 are shown in a “concave” or arced position relative to one another and the planar portion 170 (FIG. 7), the ramps need not angle towards a common center, and one of the lens ramps could be angled in the opposite direction from the plane of the planar portion, and the ramps would fall in roughly parallel planes (not shown).
In another embodiment (not shown), the waveguide includes a laminate of layers, one layer serving as a structural substrate, and the other (e.g. thin film SiON) serving to transmit the light, such as those disclosed in International Application No. PCT/US96/02662 (International Publication No. WO 96/26432, published Aug. 29, 1996) to the University of Utah Research Foundation. In such an embodiment, the structural substrate can be made of a plastic such as polystyrene, PMMA, polyvinyl chloride (“PVC”), polyimide, polyester, polyurethane, organically modified ceramics, polymers of diethylene glycol bisallyl carbonate, allyldiglycolcarbonate, polycarbonate, or equivalent material. The waveguide layer is preferably an optical plastic such as polystyrene, although it can be made of other suitable materials such as TiO2, a mixture of TiO2-SiO2, SiO2, ZnO, Nb2O5, Si3N4, Ta2O5, HfO2, or ZrO2. Waveguide layers such as TiO2, SiO2, or Si3N4 can be deposited by plasma chemical vapor deposition (“PVCD”), plasma impulse chemical vapor deposition (“PICVD”) process, or the like.
A gasket 162 is preferably seated between waveguide 164 and flow cell top 100 (
Upon assembly of the biosensor, in the reservoirs 102-106, the first planar surface 182 of the waveguide 164 constitutes a floor or ceiling (
The gasket 162 is preferably made of a semi-rigid material having an index of refraction less than that of the waveguide material in the wavelength range of the ceiling light. For best results, it is believed that the index of refraction of the gasket material should be as low as possible compared to that of the waveguide. For a waveguide made of quartz or glass, the index of refraction would typically be from about 1.46 to 1.52, higher for high-lead glass. A transparent (non-pigmented) silicon rubber (siloxane polymer) with an index of refraction of 1.35-1.43 is a presently preferred material for gasket 162. TEFLON or TEFLON-type materials such as PTFE (polytetrafluoroethylene) or FEP (fluorinated ethylene propylene) have indices of refraction of around 1.34-1.35, and may also be suitable for use as layer 196.
The other portion 198 of the gasket may be formed of an opaque (e.g. red or black) neoprene or silicon rubber material which is preferably biologically inert although due to the metal walls, it need not be.
D. The Flow Cell Assembly
As depicted in
The flow cell assembly can also includes means for associating the flow cell top 100 wit the flow cell bottom 186 thus sandwiching the gasket 162 and waveguide 162 therebetween. The depicted means for doing so are threaded clamping bolts 204, 206 which interact with correspondingly threaded holes 208, 210 in the flow cell bottom 186. Of course however, equivalent means such as screws, nuts and bolts, clamps, snap fits, and the like could alternatively be used.
A waveguide registration plate 212 is shown associated with the flow cell bottom 186 (FIG. 4). The waveguide is reproducibly positioned between the flow cell top and bottom when aligned with the registration plate. Also depicted is a 3-channel beam mask 214 having three apertures for receiving a light beam.
E. The Apparatus
Once the flow cell 200, gasket 162 and particular waveguide 164 have been associated with one another, the thus formed biosensor 190 may be used in an apparatus for performing immunoassays such as fluoroimmunoassays. As depicted in
The embodiment of
Light detection means, generally 230, are positioned to detect fluorescent light emitted from the biosensor 190. As more thoroughly described herein with regard to
The distance 234 between collection lens 232 and optical substrate 164 is selected as known to those skilled to maximize the collection of light emitted from the region of evanescent light penetration while at the same time imaging this light onto the face of the photodetector. The light collected by collection lens 232 is then sent to detection means 230, which responds by outputting signals reflective of the level of collected fluorescent light.
Detection means 230 may be any type of photodetector useful to detect light in the waveguide region spanning the wavelength range of the emitted fluorescence, as known in the art. However, in a preferred embodiment for simultaneous multianalyte assays, detection means 230 is an imaging-type detector providing direct imaging of each of the fluorescence signal(s) originating in the evanescent zone 236 (FIG. 9). In the apparatus of
Alternatively, detection means 230 may be a photomultiplier, a semiconductor photodiode, or an array of such detectors. In embodiments other than a CCD, an array is generally preferable to a single detector for some purposes. With an array of small detectors, the user can determine that the maximum fluorescence is being detected and is not inadvertently missed due to misalignment of the collection and detection optics. Optionally, a grating spectrograph is coupled to the CCD or other detection means to provide spectral analysis of the detected light. In that case, means are also provided to integrate the signal function around each peak to determine the total collected fluorescence from a sample. Alternatively, in an embodiment for use in a setting such as in a testing laboratory, and for which all the parameters of the assay have been standardized, the spectrograph may be replaced by a filter which passes only wavelengths in the region of tract fluorescence.
As is better seen in
When light is being propagated in the waveguide 164 and internally reflected at the surfaces 172, 174 an evanescent light field is produced having an intensity curve 230 which drops off with distance from the surface 172, as diagrammed relative to a distance axis 232 and a horizontal axis 234 (not to scale). Evanescent light intensity varies along axis 232, co-linear with distance. An excitation zone 236 is the only region of the solution in which the evanescent light intensity is sufficient to excite a significant or detectable fraction of tracer molecules 244 (not to scale). Tracer molecules 244 outside zone 236 will contribute little or no induced fluorescence. Excitation zone 236 is typically between about 1000 and 2000 Å in depth.
Capture molecules 240 are reactive with the analyte molecules 242, and may be whole antibodies, antibody fragments such as Fab′ fragments, peptides, epitopes, membrane receptors, whole antigenic molecules (haptens) or antigenic fragments, oligopeptides, oligonucleotides, mimitopes, nucleic acids and/or mixtures thereof. Capture molecules 240 may also be a receptor molecule of the kind usually found on a cell or organelle membrane and which has specificity for a desired analyte, or a portion thereof carrying the analyte-specific-binding property of the receptor.
The capture molecules 240 may be immobilized on the surface 172 by any method known in the art. However, in the preferred embodiment, the capture molecules are immobilized in a site-specific manner. As used in this application, the term “site-specific” means that specific sites on the capture molecules are involved in the coupling to the waveguide, rather than random sites as with typical prior art methods. Int'l PubI. No. 94/27137, which has been previously referenced, details methods for site-specific immobilization of capture molecules to the surface of the optical substrate by means of a protein-resistant coating on the substrate.
The waveguide can be designed so that multiple (e.g. four) different assays can be performed on the same sample. This is accomplished by immobilizing different types of capture antibodies on different regions of the waveguide, a process referred to as patterning. Three different patterning methods appear suitable for immobilizing antibodies to the polystyrene sensors—gasketed multiwell coating tray, liquid jet printing and photolithography. In the former, a machine similar to an ink jet printer is used to spray reagents onto a specific region of the waveguide; in this latter, ultraviolet light is used to photochemically cross-link antibodies to selected regions.
One immobilization chemistry is based on physical adsorption of antibodies to the waveguide. In one method, an antibody is briefly exposed to acidic conditions just prior to immobilization. It has been shown that this acid pretreatment step improves the antigen-binding capacity (AgBC) of immobilized antibodies by up to 3-fold in some cases. This immobilization chemistry is relatively simple and compatible with gasketed multi-well coating tray or liquid jet printing technology, but in some cases it exhibits a higher degree of non-specific binding than other methods.
The other two immobilization chemistries are based on a family of tri-block polymers of the form PEO-PPO-PEO, where PEO stands for poly(ethylene oxide) and PPO stands for poly(propylene oxide). These surfactants are sold under the trade name PLURONICS and come in a variety of chain lengths for both the PEO and PPO blocks. The PPO block is significantly more hydrophobic than the PEO blocks and adsorbs readily to non-polar surfaces such as polystrene, leaving the PEO blocks exposed to bulk solution. The free ends of the PEO chains exhibit high mobility, literally sweeping proteins away from the surface.
In both the second and third immobilization chemistries, the surface of the waveguide is coated with pluronics before attachment of antibodies, but the two chemistries differ in how the antibodies are attached. In the second chemistry a photochemical cross-linking agent is used to conjugate antigen-binding fragments (Fab′) to the PEO blocks, making this method suitable for patterning by photolithography. In the third chemistry, Fab′ fragments are attached to pluronics using a chemical cross-linking agent, making this method compatible with gasketed multi-well coating tray or liquid jet patterning. The photochemical cross-linking method was evaluated with two different PLURONICS (F108 & F105) and two different photochemical crosslinkers (BPM and BPIA). While acceptable levels of total antigen binding can be obtained with all four pairwise combinations, an unacceptable level of NSB may be obtained when antibodies are immobilized to F108 using the BPIA crosslinker. The other three pairwise combinations give very low levels of NSB (about 1.5% of total binding). Furthermore, the P105/BPM pair is especially good, giving an undetectable level of NSB.
In the embodiment of
In tests conducted with the point-of-care cardiovascular marker CK-MB (associated with acute myocardial infarction) on both plasma and whole blood, the results were comparable (taking into consideration diffusion and viscosity differences).
In the embodiment of the apparatus of
In another alternate embodiment, light source 216 is a laser diode emitting in the red wavelength region of 600-700 nm which is commercially available. The laser diode may provide about 12 milliwatts of power with a peak emission wavelength of about 635 nm. Laser diodes emitting at 633 nm are also available and can be used. For an embodiment using a wavelength in this region, it is necessary to use dyes such as cyanine dyes, which fluorescence can be stimulated by excitation with wavelengths in the red spectral region. An example of such a dye is the fluorescent dye CY5, available from Biological Detection Systems, Inc., Pittsburgh, Pa. (catalog no. A25000). The CY5 dye can be conjugated to the desired tracer molecule by the manufacturer's instructions and/or with a kit available from BDS. A second dye, CY7, may also be suitable. The dyes and methods for conjugating are also characterized in the paper by Southwick, P.L., et al., titled “Cyanine Dye Labelling Reagents—Carboxymethylindocyanine Succinimidyl Esters”, Cytometry 11:418-430 (1990). The use of laser diodes as a light source permits the biosensor and waveguide to be formed of plastic, which considerably reduces the expense of manufacture and facilitates the integral molding of the semi-cylindrical lens with the waveguide and reservoirs.
Different labels can be used which emit light at different wavelengths if desired. In such a circumstance, different types of capture molecules (e.g. antibodies reactive with different antigens) can be immobilized to the surface so that the waveguide can be used to detect more than one molecule to be detected. In such a case, multiple wavelengths can be detected by multiplexing the signal from the waveguide.
In the depicted embodiment, the device works as otherwise herein described, but each tracer molecule (e.g. Tracer Ab1, Tracer Ab2, Tracer Ab3, . . . Tracer Abx) is labeled with a different colored fluorophore (F1, F2, P3, . . . Fx).
The waveguide is illuminated by one or more different wavelengths of light 218 appropriate to excite all the fluorophores located within the evanescent region of the waveguide. In one configuration, the emissions from the different fluorophores are distinguished using bandpass band pass filters. Light rays 248, 249 and 250 are emitted from the respective labels on the tracer molecules. This light then passes through a lens 252 which collimates the emitted light onto a band pass filter 254 selective for the wavelength emitted by the particular tracer molecule label, in the depicted case, Tracer Ab1. A filter switching member, such as a wheel 256, houses, for example, three different band pass filters—each selective for a different fluorophore label. Thus, only the light rays 248 emitted by Tracer Ab1 pass through the filter 254. If spatial resolution is desired in addition to wavelength selection, the light 248 passing through the filter 254 passes through a second lens 258 which images the light 248 onto a spatially-resolved photodetector 260 such as a CCD or diode array. If only wavelength resolution is desired, the photodetector 260 may be a single spatially-integrating device, and lens 258 may be optionally omitted.
Alternatively, the wavelength selectivity may be accomplished by one of several means instead of a filter wheel, such as employing a diffraction grating, a prism, or an acousto-optical modulator to angularly separate the different emitted wavelengths and thus direct them to separate individual photodetector elements whose outputs are representative of the signal strengths in each wavelength band. In another arrangement which avoids the use of the rotating filter wheel, stationary beam splitters are employed to direct portions of the emitted light through stationary filters placed in front of individual photodetector elements.
Alternatively, if the excitation wavelengths of the different fluorophores are sufficiently separated without appreciable overlap, the light source may sequence in time through each excitation wavelength. The emitted light at any given time is released to the signal strength of the fluorophore set whose excitation wavelength is chosen at that particular time, and no further wavelength selective devices, such as filters, are needed.
The invention is further explained by the following illustrative example.
A waveguide with integrated lenses, such as that depicted in
A flow cell top, such as that depicted in
A gasket 162, such as that depicted in
A second member 186, such as that depicted in
A stage 202, best depicted in
The waveguide and integrated lenses of EXAMPLE I, the flow cell top of EXAMPLE II, the gasket of EXAMPLE III, the second member and registration plate of EXAMPLE IV, and the stage of EXAMPLE V were associated as in
The gasket was cut to correspond to the outside dimensions of the three reservoirs 102-106 of the flow cell top 100. The silicone rubber surface contacted the flow cell and the FEP surface contacted the waveguide when the assembly was clamped. Any flash present on the gasket which interfered with seating or which came over the top of the walls was carefully trimmed back with a razor knife (the top of the dam was exposed to the surface of the waveguide, but did not touch it; flash from the gasket can interfere with proper clamping).
The waveguide 164 was seated in the shallow depression in the second member 186. The waveguide fit into the depression with minimal lateral movement, but without compression or pinching. A small amount (e.g. less than about 0.1 mm (0.003 in.)) of lateral movement was acceptable. If pinching occurred, additional milling to the walls of the depression was necessary to allow proper seating.
To insure that the waveguide 164 was reproducibly positioned directly beneath the flow cell, it was butted up against the registration plate 214 after being seated in the secondary member 186. Contact with the registration plate 214 was only at the outermost corners of the front lens; no contact with the injection mold stub on the underside of the front lens occurred (injection mold may be designed to place resultant stub at an alternate location). The front of the second member may need to be milled to ensure the waveguide sits directly beneath the flow cell when in contact with the registration plate.
After seating the gasket into the flow cell and positioning the waveguide on the second member, the flow cell was mated with the second member by engaging the locating pins into the apertures in the flow cell. When fully engaged, but without adding additional clamping force (i.e., the gasket was not compressed), there was a 0.15 mm (0.006 in.) gap between the lands of the flow cell and the lands of the second member. When fully clamped with four thumb screws such that the lands are in contact, the gasket is compressed 0.15 mm (0.006 in.). The flow cell and second member readily separated using manual force; no sticking occurred, but a thin coat of lubricant may be used on the pins if necessary. It may be desirable to slightly countersink the press fit hole on the second member and/or the aperture on the flow cell to avoid burrs or bulges which might impair mating of the two parts.
The locating pins from the bottom of the second member readily aligned and fit into the apertures on the stage. No perceptible play existed between the parts when mated. As with the flow cell and second member fit, the second member and stage readily separated using moderate manual force.
As shown in
A. Waveguide Fabrication
Thin film channel waveguide layers of SiON were formed on grating etched quartz wafers in a manner such as that described in Walker et al. “Corning 7059, silicon oxynitride, and silicon dioxide thin-film integrated optical waveguides: In search of low-low, non-fluorescent reusable glass waveguides”, Appl.Spectrosc., 46: 1437-1441 (1992). Briefly, the wafers were introduced into a plasma impulse chemical vapor deposition (“PECFD”) chamber (Texas Instruments) operating at 300° C., 50 W, and 1.25 Torr. The gases used were SiH4, silane, nitrogen, ammonia, and nitrous oxide. SiON films were produced at a deposition rate of approximately 590 Å/minute for 25.42 min., yielding a film thickness of 1.5 μ. As described in Plowman et al., “Femtomolar sensitivity using a channel-etched thin film waveguide fluoroimmunosensor”, Biosensors & Bioelectronics, 11:149-160 (1996), nine parallel 1 mm×65 mm channels are formed when etched into the SiON from an additional layer of photoresist. The resulting waveguide wafers were diced (Disco DAD-2H/6) into three rectangular pieces measuring about 2.5 cm×7.8 cm each with three waveguiding channels (although in this EXAMPLE, grating waveguides without channels were employed).
B. Grating Fabrication
Quartz wafer substrates (Hoya, Woodcliff Lake, N.J., QZ 4W55-325-UP) measuring 100 mm in flat length and 0.5 mm in thickness were cleaned at room temp. in a 6% solution of H2O2 in H2SO4. The wafers then received a HMDS vacuum vapor prime, were spin-coated with photoresist (Shipley SNR 200, MA, USA) at a rate of 4200 rpm for 1 min. to produce a film approximately 0.7 mm thick and then soft-baked at 100° C. for 1 min. The negative resist was exposed to define 0.7 μ periodic groove patterns using ultraviolet light (248 nm KrF excimer laser, Laser Stepper GCA-ALS, Tukesbury, Mass., USA) with a chrome-quartz mask (DuPont, Kokomo, Ind., USA) at a dose of 20 mJ/cm2. The wafers received a post-exposure bake at 130° C. for 90 sec. Photoresist was developed in a solution of Shipley MF 312 (MA, USA) at a normalization of 0.17, then spun dry. A 60 sec. 100° C. post-development bake was then performed. The resulting photoresist pattern was reactive ion-etched (8110 Reactive Ion Etcher, AME, Santa Clara, Calif., USA) with O2 and CHF3 gases at an etch rate of 450 Å/min. Etch times of 13.33, 17.77 and 22.22 min. were employed to produce grating etch depths of 0.6, 0.8, and 1.0 μ giving aspect ratios of 0.8, 1.0, and 1.4, respectively. Residual photoresist was removed by O2 plasma.
When used as light couplers for thin film IOWs, the gratings were roughly half as efficient as prisms in coupling laser light (the light source being a 2 mW He-Ne laser (632.8 nm, 10 nW maximum, Melles-Groit, Uniphase, Manteca, Calif., USA). Both approaches (i.e. prism and grating) detected samples with femtomolar concentration above background.
An evanescent wave assay apparatus of the instant invention was compared with a standard ABBOTTS STAT CK-MB IMx system.
A. Clinical Samples
63 clinical samples (submitted by physicians for hospital CK-MB testing) were obtained, and were assayed for CK-MB using the ABBOTT IMx system. The values thus obtained for each clinical sample was noted. Each sample was aliquoted into 0.550 ml sample size, assigned a lot number, and stored at −20° C.
B. Instrumentation/Assay standardization
The samples were then assayed on a CK-MB system utilizing the biosensor 190 of the instant invention. CK-MB specificity was determined by spiking CK-MB stripped plasma (solid-phase absorption) with 1000 ng/ml CK-MM and CK-BB. Cross-reactivity with CK-MM was less than 0.1%. Standards were prepared by addition of a known mass quantity of recombinant CK-MB (Genzyme).
C. Monoclonal antibodies
Monoclonal antibodies were as described by J. Landenson, Clinical Chemistry, vol. 32, pp. 657-63 (1986). The capture antibody was coated onto the waveguide's surface with 2 hour incubation at room temp., at a concentration of 0.1 micromolar (diluted in PBSA buffer). After the incubation, each waveguide was washed once with PBSA, and then incubated with 1 ml of post-coating solution (0.5% bovine serum albumin (“BSA”)/0.1% trehalose/PBSA) at room temp. for 1 hour. The post-coating solution was discarded and the waveguides dried in a vacuumed desiccator for an hour.
E. Specific Performance
The correlation of an apparatus according to the instant invention with the ABBOTTIMx assay for an CK-MB assay is graphically depicted in
The preceding example was continued to further establish the utility of the system, and to incorporate more extensive studies of known interfering substances (e.g. hemoglobin and bilirubin), known CK-MB concentrations were analyzed (20 ng/ml CK-MB into human plasma), with concentrations of hemoglobin (15 mg/ml) and bilirubin (1 mg/ml) known to interfere with immunofluorescence assays. The system was still able to detect 100% of the CK-MB.
Characteristics of the described and illustrated embodiments are intended for illustrative purposes, and are not to be considered limiting or restrictive. It is to be understood that various adaptations and modifications may be made by those skilled in the art to the embodiments illustrated herein, without departing from the spirit and scope of the invention, as defined by the following claims thereof.
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US3490847 *||May 17, 1966||Jan 20, 1970||Philips Corp||Internal reflection devices,especially for use in spectroscopy|
|US4746179 *||Jun 30, 1986||May 24, 1988||Battelle Memorial Institute||Waveguide to be used as optical probe in multiple internal reflection spectroscopic analysis|
|US4818710 *||Dec 6, 1985||Apr 4, 1989||Prutec Limited||Method for optically ascertaining parameters of species in a liquid analyte|
|US4857273 *||Apr 4, 1986||Aug 15, 1989||Plessey Overseas Limited||Biosensors|
|US4980278 *||Apr 24, 1989||Dec 25, 1990||Olympus Optical Co., Ltd.||Method of effecting immunological analysis and apparatus for carrying out the same|
|US5064619 *||May 10, 1989||Nov 12, 1991||Amersham International Plc||Biological sensors|
|US5202950 *||Sep 23, 1991||Apr 13, 1993||Compaq Computer Corporation||Backlighting system with faceted light pipes|
|US5485277||Jul 26, 1994||Jan 16, 1996||Physical Optics Corporation||Surface plasmon resonance sensor and methods for the utilization thereof|
|US5512492 *||May 18, 1993||Apr 30, 1996||University Of Utah Research Foundation||Waveguide immunosensor with coating chemistry providing enhanced sensitivity|
|DE4412893A1||Apr 14, 1994||Oct 19, 1995||Cytech Biomedical Inc||Appts. for analytical examination of samples with a low bio-molecule content|
|EP0185126A1||Dec 10, 1984||Jun 25, 1986||Prutec Limited||Optical test probe and apparatus for optically determining species in solution|
|WO1994027137A2||May 18, 1994||Nov 24, 1994||Univ Utah Res Found||Apparatus and methods for multianalyte homogeneous fluoroimmunoassays|
|WO1995003538A1||Jul 18, 1994||Feb 2, 1995||Artificial Sensing Instr Asi A||Optical biosensor matrix|
|1||Supplementary European Search Report, EP 97 91 6086, dated Jan. 31, 2000.|
|2||Supplementary Partial European Search Report, EP 97 91 6086, dated Oct. 26, 1999.|
|Citing Patent||Filing date||Publication date||Applicant||Title|
|US8288157||Sep 12, 2008||Oct 16, 2012||Plc Diagnostics, Inc.||Waveguide-based optical scanning systems|
|US8300993||Nov 12, 2009||Oct 30, 2012||Mbio Diagnostics, Inc.||Waveguide with integrated lens|
|US8331751||Nov 9, 2010||Dec 11, 2012||mBio Diagnositcs, Inc.||Planar optical waveguide with core of low-index-of-refraction interrogation medium|
|US8465699||Jun 18, 2013||Pacific Biosciences Of California, Inc.||Illumination of integrated analytical systems|
|US8467061||Jun 18, 2013||Pacific Biosciences Of California, Inc.||Integrated analytical system and method|
|US8586347||Sep 15, 2011||Nov 19, 2013||Mbio Diagnostics, Inc.||System and method for detecting multiple molecules in one assay|
|US8606066||Dec 4, 2012||Dec 10, 2013||Mbio Diagnostics, Inc.||Planar optical waveguide with core of low-index-of-refraction interrogation medium|
|US8618507||May 24, 2013||Dec 31, 2013||Pacific Biosciences Of California, Inc.||Substrates, systems and methods for analyzing materials|
|US8649011||May 16, 2013||Feb 11, 2014||Pacific Biosciences Of California, Inc.||Integrated analytical system and method|
|US8675199||Apr 28, 2010||Mar 18, 2014||Plc Diagnostics, Inc.||Waveguide-based detection system with scanning light source|
|US8747751||Jun 10, 2009||Jun 10, 2014||Plc Diagnostics, Inc.||System and method for nucleic acids sequencing by phased synthesis|
|US8867038||Dec 16, 2013||Oct 21, 2014||Pacific Biosciences Of California, Inc.||Integrated analytical system and method|
|US8994946||Dec 21, 2012||Mar 31, 2015||Pacific Biosciences Of California, Inc.||Integrated analytical system and method|
|US9029802||Nov 19, 2013||May 12, 2015||Pacific Biosciences Of California, Inc.||Substrates, systems and methods for analyzing materials|
|US9157864||May 16, 2013||Oct 13, 2015||Pacific Biosciences Of California, Inc.||Illumination of integrated analytical systems|
|U.S. Classification||385/12, 385/146, 385/130, 385/33, 385/15, 422/82.11, 385/37|
|International Classification||G01N21/05, G01N33/543, G02B6/10, G01N21/64, G01N33/552, G01N21/00, G02B6/26, G01N21/03, G01N33/72, G02B6/00, G02B6/32|
|Cooperative Classification||G01N21/0303, G01N2021/058|
|Jan 7, 2008||FPAY||Fee payment|
Year of fee payment: 8
|Jan 27, 2012||FPAY||Fee payment|
Year of fee payment: 12