Patent Description:
This disclosure relates to photodetection in immunoassays and more particularly, to chemiluminescent detections systems and methods configured to carry out measurements of luminescent light emissions that emanate from a reaction vessel.

Chemiluminescence (CL) is defined as the emission of electromagnetic radiation caused by a chemical reaction to produce light. A chemiluminescence immunoassay (CLIA) is an assay that combines a chemiluminescence technique with an immunochemical reaction. For example, ATELLICA® IM <NUM>, IMMULITE® <NUM>, and ADVIA® Centaur, available from the present assignee are instruments based on chemiluminescence immunoassays.

Similar to other labeled immunoassays (e.g., RIA, FIA, ELISA), CLIA systems utilize chemical probes that can generate luminescent light emission through a chemical reaction to quantify the analyte (component of interest). In some automated immunoassay systems, a sample container (e.g., a cuvette) containing extracted components (e.g., DNA probes) extracted from a specimen (e.g., a biological specimen such as blood serum or plasma or urine) that have been labeled to form labeled components that can be positioned at a desired location with the system. Thereafter, by causing a reaction, luminescent intensity readings can be obtained of the luminescent light emissions that emanate from the labeled components.

For example, in some embodiments, acridinium ester labels are used to label components, such as antibodies, proteins, and some peptides, for example. Exposure of an acridinium ester label to an alkaline solution cleaves the ester linkage to release an unstable compound that decomposes to trigger a flash of luminescent light emitted at a well-defined wavelength peak (hereinafter emission peak). Luminescence, as defined herein, is light emission from a substance when it returns from an excited state to its ground state.

However, existing luminescent detection systems may be insufficient in some respects in as far as they produce luminescence intensity that can be insufficient. Hence, improved systems and methods for luminescence detection, such as in immunoassay systems, are desired.

<CIT> discloses a luminescence detection system comprising capillary channels of a micro-sampler for transferring a body fluid onto the test field of a test element, which produces diffusely scattered light when irradiated with light from a light source. The scattered light is registered by an image detector such as a standard CCD/CMOS image sensor chip which typically exhibits a maximum efficiency in the range of <NUM> to <NUM>. A wavelength-converting material such as a fluorescent material is provided which e.g. converts photons having a wavelength of <NUM> into photons of <NUM>, whereby the wavelength conversion increases the detection efficiency due to a better sensitivity of the image sensor chip at longer wavelengths.

The present invention relates to luminescence detection systems as well as methods of luminescence detection as set out in the appended set of claims.

Numerous other aspects are provided in accordance with these and other aspects of the disclosure. Other features and aspects of the present disclosure will become more fully apparent from the following detailed description, the claims, and the accompanying drawings.

In view of the above expressed issues and concerns, systems and methods that have relatively improved sensitivity are provided. Chemiluminescence technology in immunoassays relies on chemiluminescent tags at chemiluminescent labels that have high quantum yields at tag- and conjugate-specific emission wavelengths. Current photo multiplier tube (PMT) detectors used to read the chemiluminescence emissions have a maximum sensitivity at a specific wavelength (a maximum quantum efficiency %). Unfortunately, as the inventors have recognized, this maximum quantum efficiency may not be at the same wavelength as the highest quantum yield of chemiluminescence emissions. Thus, the sensitivity of detection of existing chemiluminescence detection systems may be lacking.

In particular, prior systems have used, for example, a blue dye label (e.g., with a nominal emission wavelength at from about <NUM> to <NUM>) as it best matches with the wavelength of maximum quantum efficiency (%) of the detector, which is at a wavelength of about <NUM>. However, the inventors have further recognized that light intensity or energy flux is proportional to photon flux, i.e., the number of photons per unit time per unit surface and not to individual photon energy. Thus, the number of photons at lower frequency (higher wavelength) will be more than that at higher frequency (lower wavelength). Thus, photon flux from using a blue dye label can be improved, for example by using a dye label having a higher luminescent emission wavelength range, such as a red dye label. However, when a red dye label is used, the quantum efficiency of the conventional sensor is reduced, as the luminescent emission wavelength moves away from the maximum quantum efficiency. In fact, the quantum sensitivity can be reduced more than a hundred fold by switching to a red dye label.

Thus, in order to improve sensitivity of detection, and in accordance with a first aspect of the disclosure, the chemiluminescent emissions should be matched with the detector's maximum sensitivity wavelength. However, this is a challenge with existing systems as blue label detectors are as stated above have relatively low photon flux. Thus, in existing luminescent detection systems there are losses of sensitivity due to the mismatch between the incident luminescent spectrum and the spectral absorption properties of the detector. Intensities for various assays are read out using the single detector, irrespective of their maximum quantum yield, thus losing intensity and impacting sensitivity for at least some wavelengths. The resulting lower sensitivity is simply tolerated in existing systems.

Thus, in accordance with one aspect, a luminescence detection system is provided that can sensitively measure luminescent assay emissions. In some embodiments, the luminescence detection system can be implemented on existing analyzers as a hardware retrofit upgrade. For example, the luminescence detection system can replace the existing luminescence detection system utilized in the analyzer. In accordance with one or more features of the disclosure, the improved luminescence detector system allows photons to pass through that are already in the maximum detection efficiency wavelength range (maximum detection window). However, photons that lie outside the maximum detection efficiency wavelength range (max detection window) are converted to fall into the maximum detection efficiency wavelength range (max detection window). Thus, minimum intensity loss can be incurred when using the conversion.

Thus in accordance with aspects of the present disclosure, higher total read-out of photoluminescent immunoassays can be achieved, resulting in higher sensitivity. Furthermore, aspects of the present disclosure allow the option to use higher quantum yield labels (e.g., red dye labels) that are currently out of range for conventional instrumentation. Additionally, aspects of the present disclosure allow for the conversion of "waste photons" into detected photons by use of a photoconversion layer (conversion layer) added to, or used in conjunction with, the photodetector. Thus, a greater range of immunoassays, with potentially a higher sensitivity, can be used with the luminescence detection systems of the present disclosure.

Thus, according to embodiments of the disclosure, luminescence detection systems are provided that can be implemented within immunoassay instruments to provide improved intensity of luminescent emission as well as improved luminescent emission detection sensitivity. Further, luminescence detection methods adapted to provide improved detection of luminescence emitted from a chemiluminescent reaction are provided.

Thus, it should be recognized that the methods and systems described herein cannot only improve the intensity of luminescent light emission, but at the same time can improve overall detection sensitivity. Thus, overall signal strength of the luminescence detection system can be dramatically improved.

Further details and examples of apparatus, systems, and methods of the disclosure are provided with reference to <FIG> herein.

<FIG> depicts an example embodiment of a luminescence detection system <NUM> that is configured and operable to carry out the method to measure intensity of luminescent light (e.g., luminescent light emissions) emanating from a chemiluminescent reaction of labeled components <NUM> provided in a solution <NUM> of a test sample contained in a sample holder <NUM> located at a sample location <NUM> within the luminescence detection system <NUM>. The solution <NUM> of test sample comprises a solution containing the labeled component <NUM> plus a solution that has been added to cause the chemiluminescent reaction with the labeled component. Thus, the sample holder <NUM> is configured to hold, in solution <NUM>, a test sample containing the labeled components <NUM>, wherein the component has been obtained from a processed biological specimen. The biological specimen can be any bio-fluid, such as blood serum, blood plasma, urine, cerebrospinal fluid, or the like. The sample holder <NUM> can be a cuvette or other vessel that is optically transparent or translucent, such as a plastic or glass. The walls of the sample holder <NUM> may be planar or can be curved or combinations thereof. The sample holder <NUM> may be provided at the sample location <NUM>, by entry through a door or lid <NUM> or other suitable introduction methods.

The dye label can be any suitable dye label that undergoes a chemiluminescent reaction and thus emits luminescent emissions <NUM> over a first wavelength range. For example, the dye label can emit luminescent emissions <NUM> over a wavelength range <NUM> from <NUM> to <NUM>, as is shown in <FIG>, for example. Further, the luminescent emissions <NUM> can have a spectral response having a luminescent peak <NUM> and a spectral distribution of luminescent intensity <NUM> about the luminescent peak <NUM>, as shown. This particular dye label has a luminescent peak <NUM> at between about <NUM> and about <NUM>. The spectral distribution of luminescent intensity <NUM> can be a non-normal distribution as shown, where greater that <NUM>% of the normalized intensity lies above the luminescent peak <NUM>.

Thus, the labeled component <NUM> undergoes a chemiluminescent reaction upon addition of a suitable agent and emits luminescent emissions <NUM> over the first wavelength range <NUM>. The labeled component can be any labeled analyte of interest, such as a labeled antibody, a labeled autoantibody, a labeled antigen, a labeled protein, a labeled DNA probe, a labeled marker, and the like. Nucleic acid probes and haptens may also be labeled. Labeling can be accomplished indirectly by binding conjugates or directly by direct enzyme conjugation. For example, acridinium esters are direct chemiluminescent labels for antibodies and DNA probes. Acridinium esters used as direct labels can be attached to the probe through a hybridization reaction. Acridinium esters can be reacted with alkaline peroxide (e.g., hydrogen peroxide) under basic conditions to yield an excited state, which emits light at a defined wavelength. Derivatives such as acridinium sulfonamide ester labels may also be used. Other types of luminophore and enzymatic labels may be used. The present method can be used for identifying and detecting concentrations of various components (e.g., antibodies, autoantibodies for diagnosis of autoimmune diseases, serum concentrations of hormones, polypeptides, drugs, vitamins, tumor markers, infectious disease markers, inflammation markers, myocardial damage markers, and the like) as a biochemical technique used in immunology.

Again referring to <FIG>, luminescence detection system <NUM> further includes a photodetector <NUM>, such as a photomultiplier tube (PMT), having a light entrance window <NUM> that is configured to receive at least a portion of the luminescent emissions <NUM> (photons) emitted from the chemiluminescent reaction. Light entrance window <NUM> may have a circular shape in plan view, and a receiving area that is sufficiently large, for example. Other shapes are possible. As shown in <FIG>, the photodetector <NUM> has a maximum detection efficiency wavelength range <NUM>. The maximum detection efficiency wavelength range <NUM> is the range over which the quantum efficiency is <NUM>% or greater, as shown for example. However, in some portions of the maximum detection efficiency wavelength range <NUM>, the quantum efficiency can be <NUM>% or more, <NUM>% or more, or even <NUM>% or more.

As can be seen from <FIG>, the maximum detection efficiency wavelength range <NUM> of the photodetector <NUM> is fairly efficient at receiving blue luminescent light emissions, which have a wavelength range from about <NUM> and <NUM>. However, blue dye labels emit comparatively low photon emission intensities, and thus cannot provide high detection levels. Red dye labels, however, have much higher photon emission intensities, but as can be seen from <FIG>, red light which has emissions at from about <NUM> to <NUM> would have quantum efficiencies that are too low, e.g., below about <NUM>%. Thus, using a red dye label with a blue-dominant detector would result in very low detection sensitivity.

However, according to various embodiments herein, as shown in <FIG> and enlarged <FIG>, a conversion coating <NUM> is provided at a location adjacent to the light entrance window <NUM>. For example, in a first embodiment, the conversion coating <NUM> can be applied directly to a substrate comprising a photocathode 118C in light entrance window <NUM> of the photodetector <NUM>.

The conversion coating <NUM> functions to convert at least some of the luminescent emissions <NUM> to incident emissions <NUM> that have been shifted in wavelength to fall within a second wavelength range <NUM> as shown in <FIG>. The second wavelength range <NUM> can be from <NUM> to <NUM> or even from <NUM> to <NUM> in some embodiments. As shown, the incident emissions <NUM> (photons) that contact the photocathode 118C of the photodetector <NUM> can have an incident peak <NUM> (<FIG>). Photocathode 118C converts incident photons into electrons.

The conversion coating <NUM> is designed so that the incident peak <NUM> falls within the maximum detection efficiency wavelength range <NUM> of the photodetector <NUM> as best shown in <FIG>. Thus, effectively, red-dominant light has been converted or shifted to blue-dominant light in this embodiment. Thus, a double advantage of higher photon emission generated via the use of red dominant light is achieved along with improved sensitivity by downshifting so that the incident peak <NUM> falls within the maximum detection efficiency wavelength range <NUM>. Maximum detection efficiency wavelength range <NUM> can be from <NUM> to <NUM>, for example. In some embodiments, it is desired that the incident peak <NUM> substantially coincides with the location of the quantum efficiency peak <NUM> within the maximum detection efficiency wavelength range <NUM>. By "substantially coincide," it is meant that the two peaks <NUM>, <NUM> differ in location by no more than <NUM>, so that maximum or near maximum detection sensitivity is achieved. The detection using the photodetector <NUM> can take place at one or more suitable time increments after introduction of the agent to cause the chemiluminescent reaction.

Again referring to <FIG>, the luminescence detection system <NUM> further includes, and may be controlled by, a suitable controller <NUM>. Controller <NUM> can include a suitable processor and memory for storing signals obtained from an output detection circuit <NUM>. The output of the output detection circuit <NUM> can be provided from an anode <NUM> at the end of a series of dynode stages <NUM>. Electrons are ejected from the surface of the photocathode 118C as a consequence of the photoelectric effect. The absorbed energy causes electron emission. These electrons are directed by the focusing electrode 118F toward the electron multiplier comprising the dynode stages <NUM>, where electrons are multiplied by the process of secondary emission. Such an arrangement of dynode stages <NUM> is able to amplify the small current emitted by the photocathode 118C, typically by a factor of one million or more. Output detection circuit <NUM> can then measure the resulting current at the cathode <NUM>, which provides an estimate of the amount of spectral luminescent emissions. Any suitable conventional output detection circuit and high voltage supply <NUM> may be used.

The photodetector <NUM> comprising a photomultiplier tube (PMT) can be constructed with an evacuated glass housing surrounding the series of dynode stages <NUM>. The conversion coating <NUM> may be designed so that the luminescent peak <NUM> is shifted via the luminescent emissions <NUM> passing through the conversion coating <NUM> to become incident emissions <NUM> colliding with the photocathode 118C. In particular, the wavelength shift may be sufficient to bring the shifted incident peak <NUM> within the maximum detection efficiency wavelength range <NUM>. In some embodiments, the shift may be <NUM> or more, <NUM> or more, or even <NUM> or more, for example.

The conversion coating <NUM> may be provided in the form of a transparent carrying matrix with suspended dye molecules or quantum dots, for example. The coating of the conversion coating <NUM> may be achieved by direct coating of the light entrance window <NUM> with the conversion coating <NUM>. Optionally, in an alternative embodiment, a transparent substrate <NUM> (e.g., window pane-like element) can include the conversion coating <NUM> applied thereon and can be installed in front of the photocathode 118C, such as by receiving the transparent substrate <NUM> in a slot <NUM>. Each of these embodiments may be retrofittable to existing PMT detectors.

The conversion coating <NUM> may be a thin <NUM> to <NUM> transparent layer provided with included conversion elements (suspended dye molecules or quantum dots). A reflective member <NUM>, such as reflective coating (e.g., a narrow-band reflecting coating) may be applied overtop of the conversion coating <NUM>. The reflective member <NUM> may have a narrow band surrounding and which may be centered on the quantum efficiency peak <NUM> (<FIG>) of the photocathode 118c. In some embodiments, the reflective member <NUM> can comprise a long pass dichroic mirror that lets luminescent emissions <NUM> over the first wavelength range <NUM> pass through and rejects at least some light outside of the first wavelength range <NUM>. This may aid in avoiding absorption in other parts of the spectrum. In further embodiments, the reflective member <NUM> comprises a universal detector that allows all light emissions through, but reflects and back-reflects incident emissions <NUM> of a second wavelength range <NUM> towards the photodetector <NUM>.

Whatever dye is chosen, and for whatever photodetector is chosen for the luminescent detection system, the luminescent emissions can be shifted in wavelength to substantially coincide with the maximum quantum efficiency range <NUM> of the photodetector <NUM>. Any suitable up conversion coating may be used.

<FIG> illustrates a flowchart depicting a method <NUM> of luminescence detection for use in, for example, immunoassay testing. The method <NUM> comprises, in block <NUM>, providing a luminesence detection system (e.g., luminesence detection system <NUM>) comprising a sample holder (e.g., sample holder <NUM>) holding, in solution (e.g., solution <NUM>), labeled components (e.g., labeled components <NUM>) from a biological sample, and a photodetector (e.g., photodetector <NUM>) having a light-receiving region (e.g., light entrance window <NUM>) and a maximum detection efficiency wavelength range (e.g., maximum detection efficiency wavelength range <NUM>), wherein a conversion coating (e.g., conversion coating <NUM>) is applied adjacent to the light-receiving region (e.g., light entrance window <NUM>). The conversion coating <NUM> can be applied directly on the face of the cathode 118C. Optionally, the conversion coating <NUM> can be applied as a coating on a transparent substrate <NUM> provided in front of the cathode 118C, such as a slide-in transparent glass panel coated with the conversion coating <NUM> shown in <FIG>.

The method <NUM> further comprises, in block <NUM>, causing a chemiluminescent reaction with the labeled components producing luminescent emissions (e.g., luminescent emissions <NUM>) over a first wavelength range, wherein the first first wavelength range may be from <NUM> to <NUM>, for example. Other suitable ranges may be implemented provided that a dye label undergoes luminescent emissions in that range.

The method <NUM> further includes, in block <NUM>, receiving and converting, with the conversion coating (e.g., conversion coating <NUM>), the luminescent emissions (e.g., luminescent emissions <NUM>) to a second wavelength range (second wavelength range <NUM>) having a incident peak (e.g., incident peak <NUM>) lying within the maximum detection efficiency wavelength range (e.g., maximum detection efficiency wavelength range <NUM>) wherein a quantum efficiency (%) is at least <NUM>% within the maximum detection efficiency wavelength range. In some embodiments, the maximum detection efficiency wavelength range is from <NUM> to <NUM>.

Claim 1:
A luminescence detection system, comprising:
a sample holder (<NUM>) configured to hold, in solution, a test sample (<NUM>) including labeled components (<NUM>), wherein the labeled components in the test sample undergo a chemiluminescent reaction and emit luminescent emissions over a first wavelength range, which range is from <NUM> to <NUM>;
a photodetector (<NUM>) having a light entrance window (<NUM>) configured to receive light emissions, the photodetector having a maximum detection efficiency wavelength range, which range is from <NUM> to <NUM>; and
a conversion member (<NUM>) provided adjacent to the light entrance window of the photodetector, wherein the conversion member operates to cause a conversion of the luminescent emissions over the first wavelength range to incident emissions of a second wavelength range, which range is from <NUM> to <NUM>, wherein an incident peak of the incident emissions falls within the maximum detection efficiency wavelength range, wherein the maximum detection efficiency wavelength range is a range of wavelengths where a quantum efficiency of the photodetector is <NUM>% or more.