This invention relates generally to devices for bio-assay, pathogen, or molecular analysis, and to systems and methods for such analysis. The invention may be employed for bio-assays such as sandwich or competitive assays, e.g. immunoassays, for example fluorescence immunoassays (FIA).
Optical systems may be used to determine the presence, concentration or quantity of an analyte in a test sample, quantitatively, semi-quantitatively or qualitatively. However, while such systems may be used successfully, they suffer from a number of disadvantages. For example, they may require the use of relatively expensive equipment in order to perform the assay which may not be appropriate for some determinations or may not be available for some tests. For example, in some cases a qualitative determination of the presence of an analyte may be sufficient whereas in other cases a quantitative determination may be necessary. Furthermore, such tests may be of limited performance leading to tests having to be repeated. In addition, there is a general desire to reduce the cost of the equipment used for the assay, and to reduce the time taken to perform the test. In other cases, it may not be desired to perform such an assay under laboratory conditions but instead it may be necessary to perform the test in the field with the minimum of equipment.
According to one embodiment, the present invention provides an assay device for enabling a test to be performed on an analyte. The device may have an application zone for receiving a quantity of the analyte, a waste zone, and a pathway for allowing passage of the analyte from the application zone to the waste zone. In one embodiment the device includes a quantity of a label material which will emit or modify light at least when activated and which will bind to the analyte. The pathway may also include a capture zone located between the application zone and the waste zone that has a quantity of capture material which is bound to the pathway and which will bind to any analyte that passes along the pathway so that the analyte will be bound to the pathway. The device has a first optical filter on a side thereof for allowing transmission of light emitted or modified by the label material and for blocking light of at least one other wavelength range in order to enable the device to be illuminated from one side thereof and for light emitted or modified by the label material located in the pathway to be detected from the opposite side thereof.
The device may be employed for performing lateral flow tests or other forms of test such as flow-through devices and tests and microfluidic tests, indeed for any test that may be used for competitive or sandwich assays.
This form of device may be employed in a number of ways. For example, it may be used together with an illumination and reading device in order to detect very small quantities of the analyte, or to perform quantitative tests on the analyte. However, in other circumstances it may be employed without a reader in order to perform a less sensitive or qualitative test simply to determine the presence or absence of an analyte.
According to another embodiment, the invention provides an arrangement that comprises the assay device in conjunction with a reader for detecting an output from the device when it is illuminated with light. The reader may comprise a light source and power source for the light source (or terminals for connection to a power source) for illuminating one surface of the assay device, and an optical detector for detecting light emitted by the device.
According to yet another embodiment, the invention provides a method of performing an assay, for example a lateral flow test, which comprises applying a quantity of analyte to the test device, causing the analyte to pass along the device so that it will be bound to the pathway by the capture material, and then illuminating the device in order to detect the presence or absence of analyte or the quantity of analyte by detecting the degree to which the light is modified by the label material. Thus the method may include applying a quantity of the analyte to the application zone and allowing the analyte to pass along the pathway to and beyond a capture zone where the pathway includes a quantity of capture material which will bind the analyte to the pathway, causing the analyte to contact a quantity of a label material which will emit or modify light at least when activated and which will bind to the analyte so that the analyte and label material will be bound to the pathway at the capture zone;
applying a quantity of a wash to the application zone in order to cause excess analyte and label material to flow along the pathway to the waste zone; and
illuminating one side of the assay device and detecting light that is emitted or modified by the label material from the opposite side of the device, the said opposite side of the device including an optical filter for allowing transmission of light emitted or modified by the label material and for blocking light of at least one other wavelength so that detection of the light will indicate the presence or quantity of the analyte.
Normally, the assay device will include a label zone where a quantity of the label material is located, the label zone preferably being located on the pathway between the application zone and the capture zone, so that on application of the analyte the analyte will contact the label material, and the analyte and label material will together pass along the pathway to the capture zone. However, in the broadest aspect of the invention, it is not necessary for the label material to be located on the pathway of the device when the assay device is supplied, and it is possible for the label material to be supplied separately and be applied to the device along with the analyte.
The present invention will now be described by way of example with reference to the accompanying drawings, in which:
The form of the assay device is shown in more detail in
A quantity of a label, which as used herein includes any label precursor, may be provided at a label and primary receptor zone 16 on one or both of the pathways at a position between the sample dispensing zone and the waste zone or wash collection reservoir so that as the analyte travels along the pathway the label will be taken up by the analyte and will travel along the pathway together with the analyte. A specific bond may be formed between the label and the analyte, for example a bond between an antibody pair, an antigen pair, a DNA bond or any other bond. In one example, where the analyte is avidin, streptavidin or neutravidin, the label material may be biotinylated so that it will form a biotin-avidin bond with the analyte. The presence of the analyte may thus be detected by subsequently observing the presence of the label. The label is described in greater detail below.
Downstream of the label and primary receptor zone along the pathway is a capture zone 18 where a quantity of capture material is located. The capture material is bound to the pathway and will bind to any of the analyte that passes along the pathway so that the analyte will be retained in one position on the pathway 10 corresponding to the capture material. In the case of a device that is used to detect avidin, streptavidin or neutravidin mentioned above, the capture material may also be biotinylated so that the analyte will be bound to the pathway by a biotin-avidin bond.
During the assay, a quantity of the analyte is dispensed into a dispensing port 20 that is located over one of the pathways above the application zone. The analyte may be dispensed by means of a pipette, dropper or other appropriate device in order to dispense a defined quantity of the analyte as shown in
A wash may then be applied to cause the analyte, control analyte and label to flow along the pathways toward the wash collection reservoir 12. The wash may be applied in any number of ways. For example it may be applied to sample dispensing port or an alternative port by means of a pipette, dropper or other device that will dispense a controlled quantity of wash into the dispensing ports 20 and 21. Alternatively, a wash dispensing reservoir 24 may be provided in the device at the end of the device opposite the wash collection reservoir, or on a side of the device. The dispensing reservoir may be in the form of a bladder, blister pack or sachet which may be punctured in order to cause the wash to flow along the pathways. In another form of arrangement where a reader is employed as described below, the reader may be designed to receive the assay device and be provided with a ridge or protuberance that will apply a stress to the wash dispensing reservoir and rupture it when the device is inserted into the reader so that the wash is applied automatically on insertion of the assay device into the reader.
Once the wash has been applied, substantially no material should be present on the pathway other than the analyte bound to the capture zone and the label that is bound to the analyte.
With many forms of capture and label arrangement the assay device may be ready to be illuminated and read as soon as the device is washed. However, in some cases a further processing step may be necessary in order to activate the label (which may be referred to herein at times as a label precursor). For example, where some forms of fluorescent label are used as described below, and in particular where fluorescein diacetate (FDA) is used, it may be necessary to hydrolyse the precursor and/or to heat it in order to release the label. This operation may be performed at any appropriate time. For example, if an acid or base hydrolysis step is necessary, the appropriate acid or base may be applied either before or after application of the analyte, for example along with the wash step. In this case, the wash in the wash reservoir may have a pH that will cause the hydrolysis so that the activation step occurs automatically with the washing step.
The assay device is now ready to be illuminated in order to detect the presence of analyte. This may be achieved by means of a reader as shown in
The assay device is preferably provided with a polarizing profile in order to ensure that the device cannot be inserted into the reader upside down or back to front. As will be appreciated, inserting the device upside down will cause any excitation wavelengths to be filtered out by the longer wavelength filter on the upper surface of the device. Such a polarizing profile may include a cut corner 29 which will cooperate with a corresponding corner in the reader. Also, a part-circular cut out 30 may be included in one end of the device to ensure which end of the device is inserted. Many other forms of polarizing profile may be employed.
In some cases, the assay device may be illuminated as soon as the pathways have been washed, but in other cases it may be necessary to wait for a period of time before illumination, in which case a timer may be provided in the reader in order to ensure that the device is illuminated and read at the appropriate time. For example, in the case of base hydrolysed FDA, it may be advantageous to wait for a period of from 100 to 500 seconds after hydrolysis before illumination.
Although the term “light” has been used herein, it will be appreciated that this is because the device is intended for reading visually. Any electromagnetic radiation may in principle be used to read the device, and the light will not necessarily be visible light, although this is preferred. The light may have components in the infrared or ultraviolet spectrum and may even have a spectrum in which the radiation is predominantly in wavelength ranges outside the visible wavelength range. However, as explained below, the light is preferably in the visible range.
The label material located in the device may be one that will emit or modify light, at least when activated, so that the light emitted from the device due to the presence of the analyte will differ from the illuminating light.
One surface of the device that receives the light only after it has passed through the label material, in this embodiment the top cover 8, is formed from a material that provides a first optical filter that will allow transmission of the light emitted or modified by the label material and will block light of at least one other wavelength range. This has the advantage that the effect of the label material is enhanced by removing at least some of the background light that is not affected by the label material.
Preferably the other surface of the device, that is to say the surface that will be illuminated by the light source during the reading stage, is also formed from a material that forms a second optical filter that has a different optical transmission characteristic from that of the first optical filter.
In the preferred form of device, the second optical filter will allow transmission of light of a shorter wavelength range than that of the first optical filter, for example the second optical filter may be a blue filter while the first optical filter may be a green filter. Especially the filters will be such that together they will block light in substantially the entire visible light range. In other words, in the preferred form of device, the long wavelength cutoff of the second filter will be substantially the same as, or in the region of the short wavelength cutoff of the first filter, so that the combination of the two filters will block substantially all visible light.
The filters provided in the top and bottom of the assay device may be formed from any appropriate material, for example glass, plastics materials, thin film materials or they may be holographic filters or interference filters. In an interference filter, a dielectric coating is deposited in layers to allow only the desired wavelengths to pass while light of other wavelengths is reflected. However, in view of the fact that the filters are provided on the assay device which will be a consumable item, the materials should be relatively inexpensive and so plastics filters are preferred.
If the label material is one such as a fluorescent or phosphorescent material which exhibits a Stokes shift between its absorption spectrum and emission spectrum, it is possible for the filters formed by both surfaces of the device to block substantially all light but allow fluorescence or phosphorescence caused by the label to be detected. For example,
The use of labels that can affect the wavelength of light such as fluorescent or phosphorescent materials together with optical filters on both sides of the assay device as described above rather than being associated with the reader has the important advantage that it is possible, at least for a number of tests (for example many qualitative tests or where a high concentration of analyte is present) to dispense with the optical reader so that it is possible simply to hold the assay device up to a source of white light, for example the sun, and observe the presence or absence of any bands on the pathway caused by the fluorescent label.
According to the broadest aspect of the invention, any of a number of label materials may be employed in the device. These may include simple coloured dyes or pigments that will affect the absorption spectrum of the analyte, but they are preferably fluorescent, phosphorescent or chemiluminescent materials. Examples of materials that may be employed as labels are disclosed in U.S. Pat. No. 7,796,266, the disclosure of which is incorporated herein by reference. In addition, the term “label” as used herein can include precursors of a label where appropriate, so that some additional step or steps may be needed before the material functions as an optical label, for example acid or base hydrolysis may be required or the application of heat or both.
According to one preferred embodiment, the label material comprises a lipid walled capsules, optionally having a polymer outer shell, containing a signal precursor. For example, the capsules may be formed from the lipid DSPE-PEG2000 Amine and sodium dodecyl sulphate (SDS) and containing fluorescein diacetate (FDA) as the signal precursor. These capsules may be activated by being placed in an activation solution having a pH of approximately 10.1 which is just below the pH value at which the FDA in this type of capsule will undergo rapid hydrolysis to fluorescein without additional heat. Such forms of label material are disclosed in international patent application No. WO 02/12888 A2, the disclosure of which is incorporated herein by reference. These capsules may release very large amounts of fluorescein when activated, with the result that assays employing these capsules can be extremely sensitive since the intensity of the fluorescent light can be many orders of magnitude above that of other fluorescent or phosphorescent materials. Indeed it is conjectured that it is this very high degree of fluorescence generated by activation of the FDA capsules when activated that enables the FDA capsules to be employed in an assay device according to the present invention employing relatively cheap and low performance optical filters located on a consumable component such as the assay device. According to U.S. Pat. No. 7,796,266 referred to above, it is a large Stokes shift, for example from 100 nm to 350 nm, that minimizes the need for expensive, high precision filters in the optical detection in order to eliminate background interference. However fluorecein employed according to the present invention has a Stokes shift of only about 25 to 28 nm.
In fact, the use of fluorescent or phosphorescent labels, and especially labels formed from the FDA capsules referred to above, can have the effect that the assay device can exhibit an absorption spectrum of the fluorescein when exposed to white light. Thus, according to yet another aspect, the method according to the invention includes the step of illuminating the assay device from one side with white light and viewing the device from the other side in order to detect absorption bands in the pathway caused by absorption of light by the fluorescent label material. In this method it is not necessary to include any second optical filter on the side of the device that is illuminated although it may be advantageous to do so in order to reduce the intensity of light passing through the assay device that is not affected by the absorption by the fluorescent or phosphorescent material, and so increase the proportion of light that is absorbed by the fluorescent or phosphorescent material.