HK1139738B - Flow sensing for determination of assay results - Google Patents

Description

Flow rate detection for determining assay results

The application is a divisional application of Chinese patent application 200410045275.3 (application date: 6/4/2004).

Technical Field

The disclosed subject matter relates to devices for reading measured values from an assay subject. In particular, it relates to electronic readers for use with assay test strips that use optical measurement methods.

Background

A variety of analytical devices are currently commercially available which are suitable for home use in testing of analytes. Unipath is inA lateral flow immunoassay device suitable for measuring the pregnancy hormone human chorionic membrane (hCG) is sold under the trade mark name and is disclosed in EP 291194.

In particular, EP291194 discloses an immunoassay device comprising: a porous carrier containing particulate labelled specific binding reagent for an analyte, which reagent is free to move in the wet state; and an unlabelled specific binding reagent for the same analyte, which reagent is immobilized in a detection zone or test zone downstream of the unlabelled specific binding reagent. A liquid sample suspected of containing the analyte is applied to the porous carrier whereupon the analyte interacts with the particulate labelled specific binding reagent to form an analyte-binding complex. The microparticle label is coloured, typically gold or a dyed polymer, for example latex or polyurethane. The complex then migrates to a detection zone where it forms another complex with the immobilised unlabelled specific binding reagent, allowing the extent of analyte present to be detected or observed. Due to the natural nature of the binding reaction taking place, it is necessary to wait a certain time after the test has started before the result can be read. This is particularly important for visual semi-quantitative type tests, whereby test zones or read lines are generated over time.

Various methods of timing the results have been proposed for use in commercial devices, including instructing the user to wait for a period of time before reading the assay results. Other methods include a signal, generated after a certain period of time, as disclosed in our co-pending application PCT/EP03/00274, which signals to the user that the result can now be read.

As a control and to ensure correct operation of the apparatus, a control zone is usually provided downstream of the measurement zone. A third binding reagent capable of binding to the labelled reagent is immobilised in the control zone so that when the analyte is not present, the user will be able to check that the test is correct. EP653625 discloses a lateral flow assay test strip for use with an assay reader to visually determine the extent of binding of particulate label. It is also known from US5580794 to provide an integrated assay device and lateral flow assay test strip in which the result is visually determined using reflectance measurements.

US5837546 discloses a method of actuating an immunoassay device by providing an additional electrode to a lateral flow carrier, detecting the presence of fluid on a test strip, and generating a signal to turn on the detection electronics. Due to the natural nature of the cross-flow type test, which requires release of the labelled particulate binding reagent, flow of liquid along the carrier (which is typically porous) and capture of the analyte in the detection zone, it is desirable to optimise the characteristics of the porous carrier.

The pore size of the support is an important factor and is preferably chosen to be 1-12 μm. The carrier is preferably nitrocellulose, the pore size of which may vary in part due to the manufacturing process. The assay device may also have a wick in liquid communication with the carrier for the liquid sample in the cartridge, and the carrier typically comprises two portions of different materials. Nitrocellulose is generally used as a carrier material for assay strips and has significant advantages over conventional strip materials such as paper because it has the natural ability to bind proteins without the need for prior sensitization. To optimize the assay, nitrocellulose is typically treated multiple times before use, including the use of blocking agents, such as polyvinyl alcohol, and the use of water-soluble glazes, such as sugars, to enhance the release of the labeling reagents.

The inventors have noted that the flow rate of liquid along the porous carrier is not the same for different tests. In some cases, the carrier has a tendency to flood, i.e., the liquid travels faster than normal along the carrier. In contrast, in some cases it is noted that the liquid is advancing along the carrier much slower than normal, i.e. the liquid is somewhat blocked. It has been found that these different types of liquid flow rate characteristics can lead to inaccurate results.

Due to the inconsistent nature of the materials used for the wick and porous membrane, the optimal time point for reading the results (after application of the liquid sample) will vary.

In order to provide an inherently more accurate and reliable device, it is desirable to provide alternative or additional technical features that enable the extent and/or speed of movement of the liquid sample along the porous carrier to be determined, and to exclude those flow rates that are determined to fall outside predetermined limits.

It is desirable to provide a method in which the optimum point in time for the reading result can be reliably and reproducibly determined.

Disclosure of Invention

In some embodiments, the present invention provides an assay device comprising a reader for use in conjunction with a lateral flow test strip which is capable of visually quantifying and/or qualitatively measuring the concentration of an analyte with high reliability and accuracy.

The present invention also provides an assay reader, particularly for use in conjunction with a lateral flow test strip, and a method of performing an assay measurement, wherein the extent and/or velocity of liquid flow along the test strip can be determined, and wherein in the event that the determination of liquid flow rate falls outside predetermined limits, the final assay result can be excluded.

In some embodiments, an assay result reading device for reading the result of an assay performed using a liquid transport carrier comprises: at least one light source capable of emitting light incident on at least one of two or more spatially separated regions on the carrier; a photodetector positioned so as to be able to monitor light emanating from both of said zones and to generate a signal indicative of the presence or absence of a liquid sample in the respective zone; and a calculation circuit that calculates a flow rate of the liquid flowing along the carrier in response to the signal, compares the calculated flow rate with upper and lower limits, and excludes the assay result if the calculated flow rate is outside the upper and lower limits.

In some embodiments, a method of assaying a liquid sample for an analyte of interest comprises: positioning a liquid transport carrier having at least two spatially separated zones relative to an assay result reader, the reader comprising a housing enclosing at least one light source and at least one photodetector, and said carrier being positioned such that said at least one light source emits light which is incident on at least one of said zones and light emanating from at least one of said zones is incident on said photodetector; applying or introducing a liquid sample to the liquid transport carrier; calculating a flow rate of the liquid sample from the signal generated by the at least one photodetector indicative of the presence or absence of the liquid sample in the respective zone; and determining whether the calculated flow rate is within a predetermined acceptable range.

The liquid transport carrier preferably comprises a porous carrier, such as a lateral flow assay test strip of the type well known to those skilled in the art. Alternatively, the liquid transport carrier may comprise a capillary filled chamber, channel or the like (as disclosed in US 6113885). The liquid transport carrier may be an integral part of the assay result reading apparatus, as disclosed in US 5580794. In such an embodiment, the combined reader/liquid transport carrier will generally be disposable. Alternatively, the liquid transport carrier may be a separate component which is typically introduced into the assay result reading device during the assay. In this latter embodiment, the liquid transport carrier (typically a lateral flow assay test strip) is typically very flexible and disposable after a single use, whereas the assay result reading device will be reusable and relatively expensive.

Drawings

FIG. 1 is a perspective view of one embodiment of an assay result reading device according to the present invention;

FIG. 2 is a simplified block diagram of some of the internal components of the embodiment of the reading device shown in FIG. 1;

figures 3-5 are graphs showing the various signals returned from different parts of a test stick inserted in the reading device of figures 1 and 2 and their variation with time.

Detailed Description

For the avoidance of doubt, it is expressly stated that features described herein as "preferred", "conveniently", "ideally" and the like may be employed in one embodiment in combination with other features, either individually or in combination, unless expressly stated otherwise.

In describing the various embodiments, the definitions of the various terms are as follows:

liquid sample: any liquid material suspected of containing the analyte of interest. Such samples may include human, animal, man-made samples. Typically, the sample is an aqueous solution or a biological fluid.

Examples of biological fluids include urine, blood, serum, plasma, saliva, interstitial fluid, and the like. Other useful samples include water, blood products, soil extracts, and the like, for conducting industrial, environmental, or food assays, as well as medical diagnostic assays. In addition, solid materials suspected of containing the analyte of interest may also be used as test samples, as long as they are processed to form a liquid medium, including further processing to release the analyte.

Any suitable analyte may be measured. Analytes of particular interest include proteins, haptens, immunoglobulins, hormones, polynucleotides, steroids, drugs, infectious disease agents (e.g., bacterial or viral origin) such as streptococcus, neisseria, and chicydia, as well as drugs of abuse, biomarkers such as cardiac markers, and the like.

Generally, the disclosed assay result reading apparatus and method are used to perform diagnostic assays, i.e. to provide information about the health condition of individual subjects of mammals, typically humans.

It is preferable to calculate the speed (rather than the range) at which the liquid sample varies along the liquid transport carrier.

For convenience, the flow rate is calculated between two zones on the liquid transport carrier such that the presence and passage of the liquid sample in a first, upstream zone is detected, and similarly the presence and passage of the liquid sample in a second, downstream zone is also detected. If the distance between the two zones is fixed and/or known, the relative or absolute flow rate of the liquid sample can be easily calculated by measuring the time elapsed between the first and second zones.

In principle, the first and second zones may be anywhere on the liquid transport carrier, for example, the first zone may be at the most upstream end and the second zone may be at the most downstream end. The distance between the two zones (and hence the transit time of the liquid sample) may be chosen to be any convenient value and may depend on the nature of the analyte to be assessed and the physical size and characteristics of the liquid transport carrier. For example, the liquid transport carrier may include one or more microfluidic channels that optionally contain one or more different microfluidic elements, such as a red blood cell separation device, a time gate, or a flow rate control device, all of which affect the transport rate of the sample. In practice, it is desirable that the separation of the two zones be at a normal flow rate, and that a sufficiently accurate flow rate be calculated within the time frame of the assay, so as not to delay the assay process or assay result determination. For detection and/or quantification assays of, for example, the pregnancy hormone hCG, the desired time is between 5 and 60 seconds.

Preferably, the presence or passage of the liquid sample at one or more additional zones on the liquid transport carrier is also detected. This allows a more accurate calculation of the flow rate. More flow rate calculation zones may be advantageous when the acceptable range of flow rates is narrow or the flow rate varies at different portions of the liquid transport carrier (e.g., where different portions have different flow rate characteristics due to the inclusion of microfluidic elements).

Furthermore, the provision of a plurality of "inspection zones" allows inspection of the progress of the liquid sample through each zone in a desired sequence, thereby alerting the user to abnormal flow patterns when a liquid sample is detected in the downstream zone before detection in a particular upstream zone. Such abnormal flow patterns can occur when a porous carrier is submerged (oversampled) with a liquid sample.

If the calculated flow rate is outside of the predetermined acceptable range, the test result may be declared invalid. Thus, the flow rate calculation can be used as a control feature. If the calculated flow rate is too high, the user may be alerted and the assay result discarded due to flooding of the porous carrier (e.g., due to oversampling, or failure of the assay device due to manufacturing defects, or damage in storage or use). Similarly, if the calculated flow rate is too low (e.g., due to undersampling), the test result may be discarded. In this way, errors due to over-sampling or under-sampling can be avoided.

In theory, any property of the liquid sample can be measured to calculate the rate and/or extent of liquid advancement, such as the capacitance, inductance, or resistance of the sample. The porous carrier or other liquid transport carrier may include a substance that undergoes a detectable change in the presence of the liquid sample. Nitrocellulose, which is commonly used as a porous carrier in lateral flow assay test strips, for example, is opaque (or substantially opaque) when dry, but its opacity decreases significantly when wetted. It is sufficient to measure or detect the change in optical reflection or transmission of the nitrated microcarrier after it has been wetted with the liquid sample to detect the rate and/or extent of development of the liquid sample.

Preferably the means for calculating the rate of development and/or extent of liquid sample application to the liquid transport carrier comprises an optical detection system. Such an optical detection system will typically generate one or more signals (preferably electrical signals) depending on the rate and/or extent of development of the liquid sample. In a preferred embodiment, a suitable optical system comprises at least two light sources and at least one photodetector, or at least one light source and at least two photodetectors, so as to be able to optically measure at least two spatially separated zones of said liquid transport carrier.

In theory, the light source may be external to the assay result reader, such as ambient light. However, this is extremely easy to introduce variations, so it is preferable that: (a) the assay result reader has at least one integral light source (LEDs are particularly advantageous in this respect); and (b) the assay result reader has a housing which substantially excludes, or at least severely limits, ambient light from entering the interior of the reading device. For this reason, the housing may be considered to be substantially ambient light excluding if less than 10%, preferably less than 5%, and most preferably less than 1% of visible light incident on the exterior surface of the device penetrates into the interior of the device. Opaque synthetic plastics materials such as polycarbonate, ABS, polystyrene, high density polyethylene, or polypropylene or polystyrene containing suitable light-blocking pigments are suitable for use in making the housing. An aperture may be provided in the outer surface of the housing which communicates with the interior space of the housing: a test strip or similar porous carrier may be inserted through the aperture to conduct an assay.

The liquid sample itself may have optical properties (e.g., color) that may interfere with the optical detection and/or monitoring of the development of the liquid sample along the liquid transport carrier. For example, blood samples absorb strongly in the wavelength range of 400nm to 600nm due to the presence of hemoglobin. Alternatively, the liquid sample is doped with a readily detectable substance (e.g., a dye, a fluorophore, etc.) prior to application to the liquid transport carrier, which does not interfere with the performance of the assay, but will facilitate detection (particularly optical detection) of the rate and/or extent of development of the liquid sample.

In another arrangement, the liquid transport carrier is provided with an easily detectable substance which is transported by the liquid sample. In this respect, dyes, fluorochromes, etc. are suitable. The readily detectable substance is preferably releasably immobilised on a porous carrier or the like so as to be released upon contact with the liquid sample. The easily detectable substance may be, for example, a colored substance that does not interfere with the assay. In a preferred embodiment, the readily detectable substance is a particulate label attached to a mobile specific binding reagent (specific binding for the analyte) and detection of the label in the detection zone constitutes a key feature of the assay.

The particulate label may be any substance suitable for the purpose described above, including coloured latex, dye sol, particulate gold (particulate gold). Alternatively, the particulate label may comprise a fluorophore which can be excited by an LED emitting radiation of a suitable wavelength.

A preferred optical detection system will include at least one light source and at least one photodetector (e.g., photodiode). Preferred light sources are LEDs. The reflected and/or transmitted light may be measured by a photodetector. In this specification, reflected light means that light from a light source is reflected from a porous carrier or other liquid-transmitting carrier onto the photodetector. In this case, the detectorTypically on the same side of the carrier as the light source. Transmitted light represents light that passes through the carrier, typically with the detector on the opposite side of the carrier from the light source. For measuring the reflection, the carrier may comprise a backing, e.g. white reflectionAnd a plastic layer. Thus, light from the light source will impinge on the carrier, a portion will be reflected from its surface and a portion will penetrate into the carrier and be reflected at any depth up to the depth at which the reflective layer is located. Thus, a reflectance type measurement will actually involve the transmission of light through at least a portion of the thickness of the porous carrier.

In one embodiment, the reader comprises a housing containing at least two light sources (e.g. LEDs) and corresponding photodetectors for receiving light from the LEDs.

One light source illuminates a first upstream zone of the liquid transport carrier and the other light source illuminates a second downstream zone of the liquid transport carrier, and the respective photodetectors detect light reflected and/or transmitted from each zone, the amount of light reflected and/or transmitted depending on whether the liquid sample (optionally together with any light-absorbing or light-emitting substance transported thereby) reaches the corresponding zone.

In a particularly preferred embodiment, the assay result reading device comprises three light sources, illuminating first, second and third zones of said liquid transport carrier, respectively, and measuring the liquid sample flow rate between at least two zones. For convenience, the zone where the measurement is made when calculating the flow rate is the same as the zone where the measurement is made when determining the result of the assay, e.g. if analyte is present in the sample, the first zone may be a zone where analyte-specific labelled binding reagent is immobilised, this zone being referred to as the test zone.

Ideally, the zone where the measurement is made in calculating the flow rate is also the zone where the control measurement is made to obtain a control value that is used to determine whether the assay process is correct. Such a region may be referred to as a control region.

Preferably there is a zone in which the flow rate is measured when calculated, and which is also the zone measured when the assay result reader is calibrated. This region is called the reference region.

Preferably the components of the assay reader used to detect and/or quantify the analyte of interest are also used to calculate the liquid flow rate. This has the advantage of being simple and economical, and is particularly desirable for disposable devices. In particular, preferred assay result readers have an optical detection system for detecting the presence and/or amount of the analyte of interest, the same optical detection system being used to calculate the flow rate measurement.

In a particularly preferred embodiment, the assay result reader obtains measurements from a control zone, a reference zone and a test zone, the control zone being located downstream of the reference zone and the reference zone being located downstream of the test zone (i.e. the reference zone is located between the control zone and the test zone). The reference region allows, among other functions, optical properties (e.g., reflectivity and/or transmissivity) of the liquid transport carrier when wetted (e.g., a wetted porous carrier). Conveniently, the results obtained from the test and control zones are normalised relative to the reference zone, so that any variation in the optical properties of the sample is taken into account and compensated for. This is particularly important when using biological samples such as urine, since the composition (e.g. concentration) of such biological samples varies greatly and thus the colour and colour depth varies.

The housing of the assay result reader typically includes an aperture to enable a test strip to be releasably inserted into and (preferably) engaged with the housing. The housing is designed such that the absolute amount of ambient light entering the reader is small. Preferably, suitable alignment and retention means are provided within the housing so that the strip remains in a fixed position when inserted. The light sources are arranged in the housing such that, when the test strip is correctly inserted, they are aligned with the respective zones to be measured.

The assay test strip may be any conventional lateral flow assay test strip, such as those disclosed in EP291194 or US 6352862. The test strip preferably comprises a porous carrier containing a labelled specific binding reagent and an unlabelled specific binding reagent. The light sources and corresponding photodetectors are preferably arranged such that, in use, light from the light sources strikes corresponding regions on the porous carrier and is reflected or transmitted to the corresponding photodetectors. The photodetector produces a current proportional to the amount of light falling on it, which then produces a voltage through a resistor. The amount of light reaching the photodetector depends on the amount of colored particle labels present and, therefore, also on the amount of analyte. In this way, the amount of analyte present in the sample can be determined. This method of optically determining the concentration of an analyte is described in more detail in EP 653625.

Alternatively, a test strip comprising a lateral flow porous carrier as disclosed in EP291194 may also be replaced by a test strip in which a binding reagent is placed in a capillary as disclosed in US 6113885.

To perform an assay measurement using an assay result reading device according to one of the preferred features, a test strip is inserted into the reader and a liquid sample is then applied to a sample receiving portion of the test strip. Alternatively, the liquid sample may be applied to the test strip before the strip is inserted into the reader. The sample moves along the porous carrier to a first zone, typically a test zone. When the sample is applied to the strip, the coloured particulate label is resuspended and moves with the liquid along the carrier. When the front end of the sample flow reaches the first region, the brightness of the light reaching the photodetector decreases as the colored particulate label absorbs a portion of the light. The change in reflected or transmitted light intensity is recorded. In practice, the amount of particulate label in the initial liquid front is greater than in the subsequent liquid. Furthermore, if a binding reaction occurs in the test zone due to the presence of the analyte, particulate label may tend to remain in the test zone. The final voltage-time curve observed will therefore depend on whether the zone concerned is a test zone, a control zone or a reference zone. For a three zone system, three voltage-time curves will be recorded, one for each zone, with a time delay between these curves due to the spatial separation of the measurement zones from each other, since the liquid front reaches the first zone in a shorter time than the second and third zones.

By analyzing the voltage-time curves of the various zones and the known distance of the zones from the zone, the liquid flow rate can be determined. With a simple algorithm, if the calculated flow rate is determined to be too low or too high, the final assay reading may be discarded.

In one exemplary embodiment, the assay result reading device will further comprise one or more of: a Central Processing Unit (CPU) or microcontroller; two or more LEDs; two or more photodetectors; a power source; and associated circuitry. The power source may comprise a battery or any other suitable power source (e.g., a photovoltaic cell). The CPU is typically programmed to determine whether the calculated speed and/or range is within predetermined limits.

Conveniently, the assay result reader will include means for displaying the assay result, which may be in the form of an audible or visible signal, for example. Preferably the device includes a visual display to display the assay result, which may be in the form of one or more LEDs or other light sources, so that the brightness of a particular light source or combination of light sources conveys the necessary information to the user. Alternatively, the device may have an alphanumeric or other display, such as an LCD. Additionally, or alternatively, to display the assay results, the device may also display or indicate to the user in other ways whether the results of a particular assay should be within a predetermined acceptable range, and whether the results of a particular assay should be discarded. If the reading device determines that a particular assay result should be discarded, it may prompt the user to re-assay. Displays suitable for displaying such information are well known to those skilled in the art and are disclosed in WO 9951989.

Examples of such applications are

Example 1

One embodiment of an assay result reading device according to the present invention is shown in fig. 1.

The reading device is approximately 12cm long and 2cm wide and is substantially finger or cigar shaped. In a preferred embodiment, the housing is no longer than about 12cm, about 2.5cm wide and 2.2cm high. However, any convenient form may be used, such as a credit card form reader. The device comprises a housing 2 made of a light-impermeable synthetic plastics material (such as polycarbonate, ABS, polystyrene, high density polyethylene, or polypropylene or polystyrene containing suitable light-blocking pigments). At one end of the reading device is a narrow slot or aperture 4 through which a test strip (not shown) can be inserted into the reader.

On its upper surface, the reader comprises two oblong holes. A screen of a liquid crystal display 6 is arranged in one of the apertures to display e.g. the assay result to the user in a qualitative or quantitative manner. The other aperture is fitted with an ejection means 8 which, when actuated, forcibly ejects the inserted assay device from the assay result reading device.

The assay device used with the reading device is a conventional lateral flow test stick, such as the form disclosed in US6,156,271, US5,504,013, EP728309 or EP 782707. The surface shape and size of the assay device and the slot of the reader into which it is inserted are such that (1) the assay device can only be inserted into the reader when the orientation is correct; (2) precise three-dimensional alignment of the reader with the inserted assay device, thereby ensuring that the assay result is correctly read.

Suitable assay device/reader device combinations exhibiting this precise three-dimensional alignment are disclosed in EP 833145.

When the assay device is correctly inserted into the reader, a switch is closed to activate the reader from a "sleep" mode, which is typically employed to reduce power consumption.

Enclosed within the reader housing (and thus not visible in fig. 1) are a number of components, shown simplified in fig. 2.

Referring to fig. 2, the reader comprises three LEDs: 10a, 10b and 10 c. When the strip is inserted into the reader, each LED is aligned with a respective zone of the test stick, LED10a is aligned with the test zone, LED10b is aligned with the reference zone, and LED10c is aligned with the control zone. The corresponding photodetector 12 detects the light reflected from each zone and generates a current whose magnitude is proportional to the amount of light incident on the photodetector 12. The current is converted to a voltage, buffered in buffer 14, and sent to analog-to-digital converter (ADC) 16. The resulting digital signal is read by the microcontroller 18.

In a simple arrangement, a separate photodetector is provided to detect from each zone (i.e. the number of photodetectors equals the number of zones measuring reflected light). The apparatus shown in fig. 2 is relatively mature and preferred. Two photodiodes 12 are provided. A photodiode detects light reflected from the test zone and a portion of light reflected from the reference zone. The other photodiode 12 detects part of the light reflected from the reference area and the light reflected from the control area. The microcontroller 18 turns on one LED10 at a time so that only one of the three zones is illuminated at any given time so that the signals produced by the light reflected by the various zones can be distinguished in time.

Figure 2 further shows, in simplified form, a switch 20 which closes when the assay device is inserted into the reader, activating the microcontroller 18. Although not shown in fig. 2, the device also includes a power source (typically one or two button cells) and an LCD device responsive to the output of the microcontroller 18.

In use, a dry test stick (i.e. before contacting the sample) is inserted into the reader, thereby closing the switch 20, activating the reader device, and the reader then performs an initial calibration. The intensity of light emitted from different LEDs is rarely the same, and as such, individual photodetectors typically do not have the same sensitivity. Since such variations can affect assay readings, an initial calibration is performed in which the microcontroller adjusts the length of time each LED is illuminated so that the signals measured from each zone (test, reference, control) are substantially equal and at the appropriate operating position in the linear region of the system response curve (so that changes in the intensity of light reflected from the various zones produce a proportional change in the signal).

After performing the initial calibration, the device performs a further fine calibration. This involves taking a measurement of the intensity of reflected light for each zone while the test bar is dry ("calibration value"), the subsequent measurements ("test values") being normalized with reference to the calibration value for the respective zone (i.e. normalized value ═ test value/calibration value).

To perform an assay, a sample receiving portion of the test stick is contacted with the liquid sample. In the case of a urine sample, the sample receiving portion may be extended into the urine stream, or the urine sample may be collected in a container, the sample receiving portion simply being immersed in the sample (for about 5-20 seconds). The sample may be taken when the test stick is inserted into the reader, or alternatively, the test stick may be removed from the reader for sampling and reinserted into the reader.

The intensity of light reflected from one or more (preferably all three) zones is then initially measured, typically after a specified time interval following insertion of the test stick into the reader. Preferably, the measurements are taken at regular intervals (e.g., 1-10 second intervals, preferably 1-5 second intervals). The measurement is made as a sequence of multiple readings over a short (10 milliseconds or less) period of time, thereby minimising the effect of changes in the intensity of ambient yellow which may enter the interior of the reader.

FIG. 3 is a graph showing the intensity of reflected light (arbitrary value) detected from each of three zones versus time using a sample that does not contain the analyte of interest. The curves of the test zones are indicated by cross-reference, the reference zones by circles and the control zones by a.

Considering the test zone profile, there is an initial lag phase in which the liquid sample moves along the porous carrier. During this phase, the light reflected by the test area is substantially constant. When the sample reaches the test area, the amount of reflected light decreases dramatically. This is primarily due to the absorption of light by the coloured labels transported by the liquid sample. However, a portion of the reduction in reflected light intensity is due solely to wetting of the porous carrier of the nitration microscope, since the dried nitration microscope is more reflective.

As the liquid front moves past the test zone, the amount of reflected light begins to increase and the colored labels are transported downstream through the test zone with the sample. The reflected light intensity does not return to the original value because the nitration microscope becomes wet and because a small amount of coloured particulate label remains as the liquid advances.

The curves for the reference and control zones are substantially similar to the test zone, but they are downstream of the test zone, thus lagging further. In particular, the control zone profile does not return to the original reflected light intensity because a "control line" (i.e., the colored particulate label deposited in the control zone) is created.

FIG. 4 is substantially similar to FIG. 3 and shows a plot obtained with the percentage normalization results (i.e., the test value is divided by the calibration value and multiplied by 100). The curve is expressed as a percentage of the calibration value versus time. Figure 4 shows that normalizing the test readings relative to the initial calibration readings reduces the variation in the signals from the test, reference, and control zones (although the control zone values are still lower due to the deposition of the labeled reagent in the control zone).

To calculate the flow rate of the liquid sample along the porous carrier, the reading device of the example actually compares the normalised results obtained from the test and control zones with the results obtained from the reference zone to obtain a "relative decay in reflected light intensity" (% A).

Fig. 5 shows a typical% a curve (versus time) for a sample containing the relevant analyte of interest. A positive attenuation indicates that the corresponding region reflects less light than the reference region, and a negative attenuation indicates how much the corresponding region reflects than the reference region.

Referring to the% a curve of the test zone, it is apparent that the test zone signal first decays significantly (relative to the reference zone) when the liquid sample (with the colored particulate label) reaches the test zone but not the reference zone. After about 35 seconds, the liquid sample starts to reach the reference zone, causing a sudden drop in the relative attenuation of the test zone. After about 40 seconds, the liquid front starts to leave the reference zone, resulting in an increase in the reflectivity of the reference zone and thus in an increase in the relative attenuation of the test zone. This stabilizes and eventually reaches a plateau, at just less than 30% positive decay, and the test zone acquires some colored particulate label due to the presence of the analyte of interest in the sample.

Considering the curve of the control zone, it is apparent that there is initially a sharp drop (negative decay) because the liquid sample reaches the reference zone before the control zone. The relative negative decay of the signal from the control zone begins to return to zero when the liquid sample begins to leave the reference zone before the control zone, and becomes positive when the liquid sample reaches the control zone, and reaches a plateau value of about 15%, since the deposition of labeled reagent in the control zone provides a positive control result.

Although the reader exemplified herein compares the test zone results with the reference zone results, the test zone results may also be compared with the control zone results.

Generally, the flow rate is calculated by detecting a change in the intensity of reflected light associated with the arrival of the liquid sample at a particular zone and determining the time elapsed for the liquid sample to reach each zone. More precisely, the flow rate is calculated as described below.

The signal measurements for all three zones are independent of the position of the liquid on the strip.

The attenuation of the signal at the test zone is measured relative to the attenuation of the signal at the reference zone. When the liquid front reaches the test zone, the signal attenuation will vary with respect to the reference zone, since the liquid front has not yet reached the reference zone (which is located downstream of the test zone). Timing begins when the signal attenuation of the test region relative to the reference region is greater than 10%. It should be noted that 10% represents confidence, including any margin of error added to the measurement readings, which error itself depends on various measurement parameters, such as the test strip, optics. Which may vary and be selected to any convenient value.

The liquid then proceeds to the reference zone and when the signal attenuation of the control zone relative to the reference zone is greater than minus 10% (-10%), the device considers that the liquid has reached the control zone (a negative value indicates that the control zone is downstream of the test zone). When the signal attenuation of the control region relative to the reference region is greater than (i.e., more positive) zero, the device determines that liquid has reached the control region. In this way, the time measured by the device does not have to correspond exactly to the time at which the liquid reaches the respective zone.

Although in this example the reader measures the speed at which liquid passes between the test and control zones, it measures relative to the signal obtained from the reference zone. The time of arrival of the liquid at the test and control zones can be judged in absolute terms (i.e. not by measurement relative to the reference zone).

The reader is also programmed to declare an assay result invalid if the liquid sample is detected in the control zone before the reference zone, since this indicates that the liquid sample has travelled through an abnormal path.

Example 2

A set of optics is used to determine the signal and flow rate. The maximum and minimum flow rates were set to 5 and 40s, respectively. Thus, any sample that passes over 40s is discarded due to being too slow (possibly due to undersampling), and any sample that is faster than 5s is discarded due to being too fast. The flow rate may be affected by a variety of factors, including porosity, distance between the control and test zones, and any chemical properties within the porous test strip that may alter the flow rate.

The timing is determined and set to zero when the fluid reaches the test line. A timer is then set and the time for the liquid to reach the control line is measured. As a further control check, the device monitors the passage of liquid through the reference zone. As a further control feature, the device also monitors the passage of liquid through the test, reference and control zones in sequence before considering the flow rate measurement as authentic, even if it meets the flow rate range of 5-40 s.

Of course, in other embodiments, the upper and lower flow rates may be set to a variety of different values depending on the particular properties of the test liquid and/or the factors described above.

Claims (20)

1. An assay result reading apparatus for reading the result of an assay performed using a liquid transport carrier, the apparatus comprising:

at least one light source capable of emitting light incident on at least one of two or more spatially separated regions on the carrier;

at least one photodetector positioned so as to be able to detect light emanating from each of the two said zones and to generate a signal indicative of the presence or absence of the liquid sample in the respective zone; and

a calculation circuit responsive to said signal for calculating the flow rate of the liquid sample along said carrier between said two zones;

it is determined whether the calculated flow rate is outside acceptable predetermined limits.

2. An assay result reading apparatus for reading the result of an assay performed using a liquid transport carrier, the apparatus comprising:

at least one light source capable of emitting light incident on at least one of two or more spatially separated regions on the carrier;

at least one photodetector positioned so as to be able to detect light emanating from each of the two said zones and to generate a signal indicative of the presence or absence of the liquid sample in the respective zone; and

determining the time for the liquid sample to flow along the carrier between the two zones;

it is determined whether the measured time is outside acceptable predetermined limits.

3. The apparatus of claim 1, wherein the computational circuitry further comprises comparing the calculated flow rate to a predetermined limit and discarding the assay result if the calculated flow rate is outside an acceptable predetermined limit.

4. The device of claim 2, wherein said device further comprises comparing said measured time to a predetermined acceptable limit, and discarding said assay result if said measured time is outside of said acceptable predetermined limit.

5. The apparatus of one of claims 1 to 4, wherein the at least one light source comprises a light emitting diode.

6. The device of one of claims 1 to 4, wherein the at least one photodetector comprises a photodiode.

7. The apparatus of claim 4, wherein the at least one light source is at least two light sources and the at least one photodetector is at least two photodetectors.

8. The apparatus of claim 7, wherein:

the first light source is capable of emitting light incident on one of two or more spatially separated regions on the carrier and the first photodetector detects light emanating from that region; and is

The second light source is capable of emitting light incident on another one of the two or more spatially separated regions on the carrier, and the second photodetector detects light emanating from the other region.

9. The device of any of claims 1-4, 7-8, wherein said signal indicative of the presence or absence of a liquid sample in a zone is calculated from the optical reflectivity, transmissivity, or both of said carrier.

10. The apparatus of claim 4, further comprising a housing enclosing the at least one light source and at least one photodetector.

11. The device of claim 10, wherein the housing is no greater than about 12 centimeters long, about 2.5 centimeters wide, and about 2.2 centimeters high.

12. The apparatus of claim 10, wherein the at least one light source and at least one photodetector are disposed within an area no greater than about 1 square centimeter.

13. The apparatus of claim 12, wherein the at least one light source and at least one photodetector are disposed within an area no greater than about 0.7 square centimeters.

14. The device of any of claims 1-4, wherein the signal generated by the at least one photodetector is indicative of the amount of analyte present in a zone.

15. An assay result reading apparatus for reading the result of an assay performed using a liquid transport carrier, the apparatus comprising:

at least one light source capable of emitting light incident on at least one of two or more spatially separated regions on the carrier;

at least one photodetector positioned so as to be able to detect light emanating from each of the two said zones and to generate a signal indicative of the presence or absence of the liquid sample in the respective zone; and

determining the time for the liquid sample to flow along at least two zones on the carrier;

it is determined whether the measured time is above a predetermined upper limit or below a predetermined lower limit.

16. The device of claim 15, wherein said device further comprises comparing said measured time to a predetermined upper limit or a predetermined lower limit and discarding said assay result if said measured time is above said predetermined upper limit or below said predetermined lower limit.

17. The device according to any one of claims 1-4 or 15-16, wherein the liquid sample is one of urine, saliva, serum or plasma.

18. An apparatus according to any one of claims 1 to 4 or 15 to 16 wherein the liquid transport carrier is a porous carrier.

19. The device of any one of claims 1-4 or 15-16, wherein said device further comprises a visual display to display the assay result.

20. The device of any of claims 1-4 or 15-16, wherein the device further comprises a plastic housing enclosing the at least one light source and the at least one photodetector.