Patent Application
20060128034
Rapid diagnostic test kits are currently available for testing for a wide variety of medical and environmental conditions. Commonly, such test kits employ an analyte-specific binding assay to detect or measure a specific environmentally or biologically relevant compound such as a hormone, a metabolite, a toxin, or a pathogen-derived antigen.
A convenient structure for performing a binding assay is a “lateral flow” strip such as test strip 100 illustrated in
An advantage of test strip 100 and of a lateral flow immunoassay generally is the ease of the testing procedure and the rapid availability of test results. In particular, a user simply applies a liquid sample such as blood, urine, or saliva to sample receiving zone 110. Capillary action then draws the liquid sample downstream into labeling zone 120, which contains a substance for indirect labeling of a target analyte. For medical testing, the labeling substances are generally immunoglobulin with attached dye molecules but alternatively may be a non-immunoglobulin labeled compound that specifically binds the target analyte.
The sample flows from labeling zone 120 into capture zone 130 where the sample contacts a test region or stripe 132 containing an immobilized compound capable of specifically binding the labeled target analyte or a complex that the analyte and labeling substance form. As a specific example, analyte-specific immunoglobulins can be immobilized in capture zone 130. Labeled target analytes bind the immobilized immunoglobulins, so that test stripe 132 retains the labeled analytes. The presence of the labeled analyte in the sample generally results in a visually detectable coloring in test stripe 132 that appears within minutes of starting the test.
A control stripe 134 in capture zone 130 is useful for indicating that a procedure has been performed. Control stripe 134 is downstream of test stripe 132 and operates to bind and retain the labeling substance. Visible coloring of control stripe 134 indicates the presence of the labeling substance resulting from the liquid sample flowing through capture zone 130. When the target analyte is not present in the sample, test stripe 132 shows no visible coloring, but the accumulation of the labeling substance in control stripe 134 indicates that the sample has flown through capture zone 130. Absorbent zone 140 then captures any excess sample.
One problem with these immunoassay procedures is the difficulty in providing quantitative measurements. In particular, a quantitative measurement may require determining the number of labeled complexes bound in test stripe 132. Measuring equipment for such determinations can be expensive and is vulnerable to contamination since capture zone 120, which contains the sample, is generally exposed for measurement. Further, the intensity of dyes used in the test typically degrade very rapidly (e.g., within minutes or hours) when exposed to light, so that quantitative measurements based on the intensity of color must somehow account for dye degradation. On the other hand, a home user of a single-use rapid diagnostic test kit may have difficulty interpreting a test result from the color or shade of test stripe 132, particularly since dye intensity declines within minutes.
Another testing technology, which is generally performed in laboratories, simultaneously subjects a sample to a panel of tests. For this type of testing, portions of a sample can be applied to separate test solutions. Each test solution generally contains a labeled compound that specifically binds a target analyte associated with the test being performed. Conventionally, the tests are separate because the labeled compounds that bind different target analytes are typically difficult to distinguish if combined in the same solution.
U.S. Pat. No. 6,630,307, entitled “Method of Detecting an Analyte in a Sample Using Semiconductor Nanocrystals as a Detectable Label,” describes a process that labels binding compounds for different target analytes with different types of semiconductor nanocrystals or quantum dots. The different types of nanocrystals when exposed to a suitable wavelength of light fluoresce to produce light of different wavelengths. Accordingly, binding compounds labeled with different combinations of quantum dots can be distinguished by spectral analysis of the fluorescent light emitted from the quantum dots.
In accordance with an aspect of the invention, a rapid diagnostic test system employs a labeling substance that attaches a quantum dot to a target analyte. When a detection zone that binds the labeled target analyte is illuminated, the quantum dots in the labeling substance fluoresce and emit a relatively bright light with a stable wavelength. The intensity of the fluorescent light from the quantum dots generally depends on and indicates the number of target analytes that are bound in the detection zone of the test system. A measurement of the light emitted at the wavelength associated with the quantum dots can thus provide a quantitative measurement of the concentration of a target analyte. In accordance with a further aspect of the invention, the illumination that causes the quantum dots to fluoresce stops before the measurement of the fluorescent light. The delay between stopping the illumination and measuring light intensity can be selected according to the persistence of fluorescence from the quantum dots and other materials in the test system. Fluorescence from other materials (e.g., typical organic materials) in the test system generally declines more rapidly than does the fluorescence from the quantum dots. Accordingly, delaying measurement after shutting off the source of illumination can provide a high signal-to-noise ratio, accurate quantitative measurements, and high sensitivity.
In accordance with a further aspect of the invention, a decay time of the fluorescence of quantum dots, which is long enough that a gated measurement provides a high signal to noise ratio, is sufficiently short for rapid repetition of gated measurements. The repetitions of the gated measurements provide statistics for better measurement accuracy without requiring an unacceptably long measurement time.
One specific embodiment of the invention is a rapid diagnostic test system including a light source, a photodetector, and a control system. The light source illuminates a medium such as a lateral-flow strip containing a sample under test and a labeling substance that binds a quantum dot to a target analyte. The photodetector measures light from a test area of the medium. The control system is coupled to the light source and the photodetector and executes a measurement processes including processing a measurement signal from the photodetector that indicates a light intensity after the light source has been off for a time.
Another specific embodiment of the invention is a process for rapid diagnostic testing. The process includes: applying a sample to a medium containing a labeling substance that binds a quantum dot to a target analyte; illuminating a portion of the medium with light capable of causing the quantum dot to fluoresce; stopping the illumination of the portion of the medium; measuring light from the portion of the medium after the illumination remains stopped for a delay time; and determining a test result from the measuring of the light.
Use of the same reference symbols in different figures indicates similar or identical items.
In accordance with an aspect of the invention, a rapid diagnostic test system employs quantum dots as labels for a target analyte and gated measurements of the fluorescent light from quantum dots for generation of quantitative or qualitative test results. The test system can include a light source that illuminates a test area with light of the proper wavelength to cause fluorescence of the quantum dots, a photodetector such as a photodiode or a sensor array that measures the resulting fluorescent light to detect the target analyte, and a control system that shuts off the light source and waits a prescribed interval before using the photodetector or sensor array for measurement of the fluorescent light from the quantum dots.
Case 210 can be made of plastic or other material suitable for safely containing the liquid sample being analyzed. In the illustrated embodiment, case 210 has an opening through which a portion of test strip 220 extends for application of the sample to a sample-receiving zone 222 of test strip 220. Alternatively, test strip 220 can be enclosed in case 210, for example, when application of the sample to test strip 220 is through an opening in case 210.
Test strip 220 can be substantially identical to a conventional test strip such as test strip 100 described above in regard to
Light source 250 in circuit 240 illuminates test stripe 226 and control stripe 228 to cause quantum dots in stripes 226 and 228 to fluoresce. Light source 250 is preferably a light emitting diode (LED) or a laser diode that emits light of a suitable frequency for illumination of test stripe 226 or control stripe 228. Generally, the quantum dots fluoresce under a high frequency (or short wavelength) light, e.g., blue to ultraviolet light, and the fluorescent light has a lower frequency (or a longer wavelength) than the light from light source 250. Test system 200 and particularly test strip 220 generally includes other materials such as nitrocellulose or other organic materials that also fluoresces when exposed to light from light source 250. These materials thus produce background fluorescent light that can complicate precise measurement of the fluorescent light from the quantum dots.
Photodetectors 256 and 258 are in the respective paths of light emitted from test stripe 226 and control stripe 228 and measure the fluorescent light from the respective stripes 226 and 228. A baffle or other light directing structure (not shown) can be used to direct light from test stripe 226 to photodetector 256 and light from control strip 228 to photodetector 258. Photodetectors 256 and 258 optionally have respective color filters 257 and 259 that transmit light of the frequency associated with the fluorescent light from the quantum dots and block other frequencies of light.
In one embodiment of the invention, the labeling substance can include two types of quantum dots. One of the types of quantum dots emits a first wavelength of light and is attached to a substance that binds to the target analyte and to test stripe 226. The other type of quantum dot emits light of a second wavelength and binds to control stripe 228. Color filters 257 and 259 can then be designed so that photodetector 256 measures fluorescent light from the type of quantum dot that test stripe 226 traps when the target analyte is present and photodetector 258 measures fluorescent light from the type of quantum dot that control stripe 228 traps once the flow of liquid has reached control stripe 228.
Quantum dots provide fluorescent light that is generally persistent for a relatively long time after illumination of the quantum dots has stopped.
An optimal delay between turning off source (step 420) and measuring the intensity of the fluorescent light from the quantum dots (step 440) will generally depend on all of the sources of noise in the test system. A long delay increases the ratio of fluorescent light from quantum dots to the fluorescent light from other sources as described above, but as the intensity of fluorescent light from quantum dots drops other sources of noise such as light leakage (e.g., into case 210 of system 200) and electronic signal noise become more important. An optimal delay for a specific test kit can be determined that will provide the highest signal-to-noise ratio when accounting for all sources of noise. In a typical embodiment of system 200 including color filters 257 and 259 on photodetectors 256 and 258, a delay time of between about 5 ns and about 100 ns may be optimal. However, delays of up to 200 ns or even up to 500 ns could also be used with quantum dots having longer half-lives for fluorescence.
For system 200, detectors 256 and 258 perform the light intensity measurement (step 440), and the intensity of the fluorescent light from each stripe 256 or 258 is proportional to or otherwise dependent on the number of quantum dots in the corresponding stripe 226 or 228. These intensity measurements thus provide a quantitative indication of the concentration of the target analyte. Step 450 can thus use the intensity measurements to determine a test result that is output from the test system. To implement step 450 in system 200, control unit 254 can be a standard microcontroller or microprocessor with an analog-to-digital converter that receives electrical signals from detectors 256 and 258. The electrical signals from detectors 256 and 258 respectively indicate the measured intensities from stripes 226 and 228 and can be converted to digital values. Control unit 254 can subsequently process the digital measurements and then operate an output system as required to indicate test results.
Optionally, a decision step 460 determines whether the process of steps 410 to 450 is repeated to generate multiple digital measurements of fluorescent light intensity. Processing of the multiple measurements can provide more sensitive/accurate quantitative measurements. One advantage of quantum dots is that a typical quantum dot can be excited and measured more than 106 times per second, allowing gated measurements to be performed at frequencies of about 1 MHz or more. For example, a measurement frequency of about 200 MHz can be achieved when the combined excitation and delay time is 5 ns. In contrast, phosphors having a half life for fluorescent light of about 1 ms or longer can similarly be used with gated measurement to reduce background fluorescence but can only be excited about 100 times per second, assuming each excitation and delayed measurement together take about 100 times the half life of the emitting material. As a result, quantum dots can be much more “luminescent” or show increased sensitivity by factors from about 100 to 10,000 times the sensitivity of a similar system using phosphors.
The output system of system 200 shown in
LED lights 261 and 263 can alternatively be replaced with other types of interfaces. For example, an alphanumeric display can provide a numerical test result based on the measurements of fluorescent light from test stripe 226. Such display could also be used in conjunction with LEDs such as illustrated in
Gated measurements can also be used in test systems employing multiple species of quantum dots.
Test strip 620 can be substantially identical to test strip 220, which is described above, but test strip 620 includes multiple labeling substances containing respective species of quantum dots. Each labeling substance binds a corresponding type of quantum dot to a corresponding target analyte. The quantum dots for different labeling substances preferably produce fluorescent light having different characteristic wavelengths (e.g., 525 nm, 595 nm, and 655 nm). Suitable quantum dots having different fluorescent frequencies and biological coatings suitable for binding to analyte-specific immunoglobulins are commercially available from Quantum Dot, Inc. Test strip 620 includes a test stripe 626 that is treated to bind to and immobilize the different complexes including the target analytes and respective labeling substances. Testing for multiple analytes in the same test structure is particularly desirable for cholesterol or cardiac panel test system that measures multiple factors.
Light source 250 illuminates test stripe 626 with light of a wavelength that causes all of the different quantum dots to fluoresce. Fluorescent light from test strip 626 will thus contain fluorescent light of different wavelengths if more than one of the target analytes are present in test strip 626. When light source 250 is turned off, the intensity of fluorescent light falls exponentially as described above, so that after a short delay time, (e.g., about 50 ns to 1 μs) the fluorescent light is almost entirely from the quantum dots.
Optical system 630 separates the different wavelengths of light and focuses each of the different wavelengths on a corresponding photodetector 642, 643, or 644. Photodetectors 642, 643, and 644, which can further include appropriate color filters, thus provide separate electrical signals indicating the number of quantum dots of the respective types in test stripe 626 and therefore indicate concentrations of the respective target analytes. Control circuit 254 can then provide the test results to a user or a separate device as described above.
Optical system 630 in
Light reflected from filter 736 is incident on filter 737. Filter 737 is designed to reflect light of the wavelength corresponding to detector 643 and transmit other wavelengths. Lens 733 focuses the light reflected from filter 737 onto the photosensitive area of detector 643. Light transmitted through filter 737 is incident of filter 738, which is designed to reflect light of the wavelength corresponding to detector 644 and transmit the unwanted wavelengths. Lens 734 focuses the light reflected from filter film 738 onto the photosensitive area of detector 644.
Optical systems 630 and 730 merely provide illustrative examples of optical system using diffractive elements or thin-film filters for separating different wavelengths of light for measurements. Optical systems using other techniques (e.g., a chromatic prism) could also be employed to separate or filter the fluorescent light of different frequencies. The characteristics and geometry of such optical systems will generally depend on the number of different types of quantum dots used and the wavelengths of the fluorescent light.
Although the invention has been described with reference to particular embodiments, the description is only an example of the invention's application and should not be taken as a limitation. Various adaptations and combinations of features of the embodiments disclosed are within the scope of the invention as defined by the following claims.
