WO2001076743A2

WO2001076743A2 - Fluid analyte detection based on flow rate or viscosity measurement

Info

Publication number: WO2001076743A2
Application number: PCT/GB2001/001650
Authority: WO
Inventors: Elizabeth Anne Howlett Hall; Ian Stuart Harding
Original assignee: Cambridge University Technical Services Limited
Priority date: 2000-04-12
Filing date: 2001-04-12
Publication date: 2001-10-18
Also published as: AU2001248542A1; WO2001076743A3; GB0008950D0

Abstract

A sensor for quantifying the flow rate or viscosity of a fluid sample, or the concentration of an analyte within that fluid sample. The sensor comprises a chamber for holding a fluid sample and guiding it through a channel containing a detection means. The chamber has at least one and preferably two changes in a diameter and the flow of the sample from the chamber thereby causes detectable changes in the readings of the detection means. Given the known chamber dimensions, this enables flow rate and viscosity of a fluid sample to be calculated. This can then be used to calculate the concentration of an analyte, directly or indirectly, despite any flow rate dependency of the detection means.

Description

APPARATUS AND METHOD FOR ASSAY OF A FLUID ANALYTE

The present invention relates to the field of analysis systems able to measure the flow rate or viscosity of a fluid sample. In some embodiments, the flow rate or viscosity is used in calculating the concentration of an analyte in a fluid sample. The invention relates in particular to the design of a flow channel and sample chamber attached thereto for use as or in an analytical measurement system.

A general trend in the application of analytical methods outside a traditional laboratory environment is to look for solutions which allow the integration of all analytical steps, requiring minimum reagent and material cost, highest device manufacturing yield and ease of user operation. As a result of this a plethora of designs and solutions have been perceived; some producing complete systems, while others have addressed single components thereof. The biosensor (e.g. the glucose electrode invented by Clark & Lyons) is an example of an analytical system with integrated reagents; whereas El A, enzyme immuno assay (specifically EL1SA and related systems), is an embodiment which demonstrates many of the principles of a total analytical procedure but generally requires user intervention or laboratory based automation to measure, deliver and remove reagents for the successive steps, prior to signal monitoring. The concept of a total analytical system can be realised in the laboratory through fluid delivery systems, valves, pumps and columns, eg FIA (flow injection analysis), whereby detection methodology as diverse as electrochemistry and optics can be incorporated. Miniaturized total analysis systems (μTAS), where all the sample-handling steps necessary to detect and quantify a range of analytes are performed extremely close to the place of measurement" offer many advantages. These include the cheapness and replaceability of components which can be fabricated in large-volume batches, the low operational costs associated with low consumption of chemicals, analytes and power¹", and the promise of a 'black box' unit which will give reliable analyses in the hands of unskilled operators. Another advantage of such a miniaturized system is its potential to be made into a portable device.

Movement of analytes within miniaturized systems provides challenges because of the high frictional forces that oppose movement of fluids through narrow channels. Miniature liquid-moving systems have been formed using micropumps^{,v v}'^v' or osmotic pumping"¹'"" but it is quite common just to use a separate pumping system. However, all these methods compromise the advantages of the μTAS approach, either by increasing the fabrication complexity and hence cost or by reducing the portability of the final sensor.

Capillary-fill is a known means of achieving sample and/or reagent delivery; this can achieve integrated sample collecting and measurement and consists of a device possessing a cavity or cavities having dimensions small enough to enable sample liquid to be drawn into the cavity by capillary action.

Alternatively, one recent development has been the 'fill and flow' strategy¹", where an increase in cavity or channel scale compared with μTAS has relieved the constraints of high frictional forces such that a sample can drive itself through the device by its own weight, interacting with analytical reagents en route. Once the sample has been added to the reservoir above the channel or cavity, the flow rate is determined by head height, which will vary as the sample drains through the device. Sensors (eg electrodes) are placed in the channel or cavity, which give the monitored signal as the sample passes. Due to the moderate increase in scale of the system such a strategy completely removes the need for an associated pumping system but if the signal from sensors used in the channel are susceptible to flow, this parameter must also be measured or some other flow marker introduced for the sensor signal to become analytically meaningful for other parameters. For example, for an amperometric electrode in a laminar flow channel,

where /ij_m is the diffusion-limiting current which is measured and which is proportional to c_m the bulk concentration of the electroactive analyte species. For a given analyte, n the number of electrons transferred, D the diffusion coefficient should be constants. W the electrode width, x_e the electrode length, h the cell half height, and of the channel width are constants which describe the channel and Vf is the volume flow rate through the channel. To obtain /, V_s must be known, but without a pumping system to control flow, this varies according to sample head-height in the reservoir above the channel and according to sample to sample variation (eg changes in viscosity). The 'fill and flow' channel has thus gained little favour in the design of analytical systems.

A first aim of the present invention is to provide an assay for the viscosity of a fluid sample or its flow rate through a sensor. The assay should ideally be carried out with minimal user intervention and better reproducibility, reliability and tolerance of sample variation than is found in the prior art.

A second aim is to provide an assay for the concentration of an analyte in a fluid sample. This assay should also ideally be carried out with minimal user intervention and better reproducibility, reliability and tolerance of sample variation than is found in the prior art. In some aspects of the present invention, the measurement of fluid flow rate/viscosity is used to achieve this second aim.

A further aim is to provide said assays without requiring pressurising or pumping technology and avoiding the cost and complexity of automatic liquid dispensing systems.

According to a first aspect of the present invention there is provided an analytical device for measuring the flow rate or viscosity of a fluid sample, the analytical device comprising a chamber and a flow rate dependent detection means, the chamber functioning to hold the sample but allow it to flow therefrom, the flow rate dependent detection means being sensitive to flow of the sample, characterized in that the chamber has at least one feature which causes a change in sample flow rate when the sample is at a location in the chamber and the flow rate dependent detection means is used to establish when the sample is at said location.

Preferably, the analytical device further comprises a channel, wherein the chamber allows the sample to flow through the channel and the flow rate dependent detection means is sensitive to the rate of sample flow through the channel.

More preferably, the sample flows under the influence of gravity. The chamber may be vertical.

Preferably, the feature which causes a change in sample flow rate when the sample is at a location is a change in the cross-sectional area of the chamber. Preferably also, the change in cross-sectional area is a discontinuity in cross-sectional area. Typically, the chamber has two or more changes in cross- sectional area. Preferably, the analytical device is adapted to calculate the flow rate or viscosity of the fluid sample from the time taken by the sample to move from one feature to a second feature.

The detection means may comprise a sensor for a component of the sample. The detection means may comprise a sensor for a component added to the sample. The detection means may comprise a sensor for matter produced responsive to a component of the sample.

The detection means comprise an electrochemical sensor and/or an optical detection means.

Preferably, the analytical device has a flow-rate restriction means. Preferably, the flow rate restriction means is a porous plug. The flow rate restriction means may comprise an electrochemical sensor.

Preferably, the analytical device has a waste collection means. The waste collection means may comprise a waste guide means and a waste collector. The waste guide means is preferably a rod.

According to a second aspect of the present invention there is provided an analytical device for measuring the concentration of an analyte in a fluid sample, the analytical device comprising a chamber and a flow rate dependent detection means, the chamber functioning to hold the sample but allow it to flow therefrom, the flow rate dependent detection means being sensitive to the rate of fluid flow, characterized in that the chamber has at least one feature which causes a change in sample flow rate when the sample is at a location and the detection means is used to establish when the sample is at said location and thereby to calculate the rate of sample flow through the chamber at a given point in time.

Preferably, the analytical device further comprises a channel, wherein fluid flows from the chamber through the channel and the flow rate dependent detection means is sensitive to the rate of fluid flow through the channel.

The flow rate dependent detection means may comprise a sensor for the analyte.

The analytical device may have a reaction zone which functions to produce chemical species in a concentration which depends on the concentration of the analyte. The reaction zone may comprise means to convert the analyte to a chemical species which can be detected by a chemical species sensor. The reaction zone may displace or produce detectable matter in response to the presence of the analyte. The reaction zone may remove chemical species from a sample in response to the presence of analyte. The reaction zone may comprise a layer of reaction component on a channel wall. The reaction zone may comprise reaction component immobilised on beads in the channel or chamber. The amount of chemical species converted, produced or displaced from the reaction zone may be independent of flow rate. The reaction zone may be selected such that its functionality is altered by the concentration of analyte. The reaction zone may comprise an enzyme which catalyses a reaction forming a chemical species, wherein the activity of the enzyme is altered by the presence of the analyte. In some embodiments the analyte may be any chemical which has an effect on the reaction zone.

The reaction zone may comprise antibody to an analyte, wherein the reaction zone carries out immunoassays including sandwich and/or competitive i munoassays.

The detection means may comprise and electrochemical and/or optical detection means. Preferably, the sample flows through under the influence of gravity. More preferably, the chamber is vertical.

The feature which causes a change in sample flow rate when the sample is at a known location may be a change in the cross-sectional area of the chamber. Preferably, the chamber comprises at least two changes in cross-sectional area. Typically, the flow rate or viscosity of the fluid sample is calculated from the time taken by the sample to move from one change to a second change. Preferably, the change in cross-sectional area is a step change.

Preferably, the analytical device has a flow-rate restriction means. Preferably, the flow rate restriction means is a porous plug. The flow rate restriction means may be an electrochemical sensor.

The analytical device preferably has a waste collection means. More preferably, the waste collection means comprises a waste guide means and a waste collector. Most preferably, the waste guide means is a rod.

The analytical device may comprise a plurality of chambers. A or each chamber may have a plurality of detection means for different analytes.

According to a third aspect of the present invention there is provided a method for measuring the flow rate or viscosity of a fluid sample, comprising the steps of:

(a) adding said fluid sample to a chamber, the chamber being adapted to allow the fluid sample to flow therefrom and comprising at least one change in cross-sectional area, (b) using a flow rate dependent detection means to make a reading sensitive to the sample flow rate, (c) using the detection means to establish when the sample is at one or more known locations with reference to the at least one change in cross-sectional area, (d) thereby calculating the flow rate and/or viscosity of the sample. Preferably, the fluid sample flows from the chamber into a channel and the flow rate dependent detection means is sensitive to the flow rate of the sample through the channel. More preferably, the sample flows under the influence of gravity. Most preferably, the chamber is vertical.

Preferably, the change in cross-sectional area is a step change in the cross-sectional area of the chamber. The chamber may comprise at least two changes in cross-sectional area.

Preferably, the flow rate or viscosity of the fluid sample is calculated from the time taken by the sample to move from one change to a second change.

The detection means may comprises an electrochemical and/or optical detection means.

Preferably, the channel has a flow-rate restriction means. More preferably, the flow rate restriction means is a porous plug.

The fluid preferably flows into a waste collection means. The waste collection means may comprise a waste guide means and a waste collector. The waste guide means preferably comprises a rod.

According to a fourth aspect of the present invention there is provided a method for measuring the concentration of an analyte in a fluid sample, comprising the steps of:

(a) adding said fluid sample to a chamber, the chamber being adapted to allow the fluid sample to flow therefrom and comprising at least one change in cross-sectional area; (b) using a flow rate dependent detection means to make a reading sensitive to the sample flow rate; (c) using the detection means to establish when the sample is at one or more known locations with reference to the at least one change in cross-sectional area; (d) thereby calculating the flow rate and/or viscosity of the sample; and (e) thereby establishing the concentration of the analyte in the sample.

Preferably, fluid flows from the chamber into a channel, wherein the flow rate dependent detection means is sensitive to the rate of fluid flow within the channel.

The flow rate dependent detection means may comprise a sensor for the analyte. The analyte may be converted to a chemical species which can be detected by the a sensor for said chemical species.

The method may further comprise the step of causing detectable matter to enter or leave the fluid sample in a concentration which depends on the concentration of the analyte. A chemical species may be displaced or produced in response to the presence of the analyte. Detectable matter may enter or leave the fluid sample in a concentration which depends on the concentration of the analyte as a result of the action of a layer of reaction component on a channel wall within the channel or chamber. Detectable matter may enter or leave the fluid sample in a concentration which depends on the concentration of the analyte as the result of the action of a component immobilised on a support within the channel or chamber. The amount of detectable matter which enters or leaves the fluid sample depending on the concentration of the analyte may be independent of flow rate. The amount of detectable matter which enters or leaves the fluid sample depending on the concentration of the analyte may be responsive to the concentration of analyte. The amount of detectable matter which enters or leaves the fluid sample depending on the concentration of the analyte may be controlled by an enzyme which catalyses a reaction forming a chemical species, wherein the activity of the enzyme is altered by the presence of the analyte. In some embodiments, the analyte may be any chemical which causes detectable matter to enter or leave the fluid sample. The method may further comprise the step of carrying out immunoassays, including sandwich or competitive immunoassays, using an antibody to the analyte.

The detection means may comprises an electrochemical and/or optical detection means. Preferably, the sample flows under the influence of gravity. More preferably, the chamber is vertical.

The change in cross-sectional area may be a step change in cross-sectional area. The chamber may have two or more changes in cross-sectional area. The method may further comprise the step of calculating the flow rate or viscosity of the fluid sample from the time taken by the sample to move from one change to a second change.

Preferably, the channel has a flow-rate restriction means. More preferably, the flow rate restriction means is a porous plug. The flow rate restriction means may comprise an electrochemical sensor.

Preferably, the fluid sample flows into a waste collection means. More preferably, the waste collection means comprises a waste guide means and a waste collector. The waste guide means is a rod.

The method may further comprise the step of using multiple chambers. More than one detection means may be used for different analytes per sample.

According to a fifth aspect of the present invention there is provided a chamber for holding a fluid sample and allow said fluid sample to drain under the influence of gravity, the chamber comprising at least one change in cross-sectional area.

Preferably, the change in cross-sectional area is a step change in cross-sectional area.

More preferably, the chamber comprises at least two changes in cross-sectional area.

According to a sixth aspect of the present invention there is provided a computer program for determining the flow rate or viscosity of a fluid sample inserted into and flowing from a chamber, the computer program comprising program instructions for a) analysing a time course of readings from a flow rate dependent detection means to determine when a fluid sample passed one or more features in the cross-section of the chamber; and

b) using the known dimensions of the chamber to thereby calculate the flow rate or viscosity of the fluid sample.

Preferably, the features are step changes in the cross-sectional area of the chamber.

More preferably, the flow rate or viscosity is calculated from the time taken for the sample to pass between two or more features.

According to a seventh aspect of the present invention there is provided a computer program for determining the concentration of an analyte in a fluid sample inserted into and flowing from a chamber, the computer program comprising program instructions for

a) analysing a time course of readings from an analyte detection means, the analyte detection means providing readings dependent on the sample flow rate, to determine when a fluid sample passed one or more features in the cross-section of the chamber,;

b) using the known dimensions of the chamber to thereby calculate the flow rate or viscosity of the fluid sample; and

c) thereby calculating the analyte concentration.

More preferably, the flow rate or viscosity is calculated from the time taken for the sample to pass between two or more features. Example embodiments of the present invention will now be illustrated with reference to the following Figures in which:

Figure 1 A and IB show a schematic illustration of an alternative apparatus including 'sample retention' in the channel design, which can be used for single assay. Figure 1 A shows a side view; Figure IB shows a top view;

Figure 2 is similar to Figure 1 except the Channel contains an electrochemical detection system. The electrodes are in contact with the fluid passing through the channel;

Figure 3 is a schematic illustration of one embodiment of the present invention which is similar to figure 2 except it includes 'sample output and collection' which can be used in a multiple sample assay system;

Figure 4 shows embodiments of Figure 3 where overall flow reduction is include by A placing a porous plug between the Chamber-reservoir and the Channel; B placing a porous plug in the outlet port; C including a rod or tube annulus to constrict the cross section of the entry to the Channel. This tube may also be used to introduce reagents or wash;

Figure 5 illustrates some combinations of application of the Chamber together with reagents;

Figure 6 illustrates a sensing area(s) within the Channel;

Figure 7 illustrates the procedure for immunoassay. Figure 7A illustrates a sandwich format; Figure 7B illustrates a competitive format;

Figure 8 shows data for hydrogen peroxide determination in the Chamber-Channel unit as illustrated in Figure 4B with a Pt working electrode; Figure 9 shows data for glucose determination with glucose oxidase immobilised on a glass bead column in a Chamber (Figure 5A) and used in a Chamber-Channel unit as illustrated in Figure 4A with a Pt working electrode;

Figure 10 shows data for determination of vanadate via enzyme inhibition of alkaline phosphatase. The enzyme was immobilised as in Figure 5A and the Channel used as in Figure 4A with a Au working electrode; and

Figure 11 obtains the viscosity of samples in the range l-20cP using the Chamber-Channel in Figure 4C with a Au working electrode.

Figures 12A to 12D illustrate alternative configurations of chamber.

This invention concerns the design of the reservoir or chamber above a flow channel and used together with the channel, the latter containing a flow sensitive sensing area or areas. The linked chamber- channel is a unit in an analytical measurement system.

The present invention uses a means for detecting and quantifying an analyte and giving a signal which is a function of sample flow rate. This flow rate dependent detection means may be flow rate dependent due to its comprising a physical or chemical sensor with flow sensitivity (eg an electrochemical or optical sensor etc). Alternatively, the flow rate dependency may arise from an analyte sensitive area (eg enzyme, antibody, analytical reagent) which engages in a reaction with a target component of the sample with reaction turnover dependent on flow. In this case, a product or other measurable parameter resulting from that reaction may be determined by a sensor which is not itself flow rate dependent and still provide a detection means which is, as a whole, flow rate dependent. For example, the flow rate dependent means may comprise a means for carrying out a flow rate dependent reaction producing a chemical which is quantified by a non flow-rate dependent sensor for that chemical.

The design of the internal profile of the chamber above the channel allows "markers" to be introduced automatically into the signal measured as the sample, which is placed in the chamber, drains into the channel. Markers are preferably discontinuities in the flow-sensitive signal which is measured at the flow rate dependent detection means placed downstream. This results from a disruption or change in the flow from the chamber caused by the shape of the feature designed in the internal profile of the chamber.

In an alternative embodiment, the markers are not single discontinuities but continuous shape elements, altering the cross-sectional area of the chamber and leading to a resulting change in the flow-sensitive signal.

The analytical measurement system disclosed herein comprises a channel with flow rate dependent detection means and a chamber as described below and functions to achieve two key related goals. These goals are, firstly, measurement of the viscosity of a fluid sample and, secondly, measurement of the concentration of an analyte in the fluid sample.

These goals are achieved by providing an analytical measurement system able to measure the flow rate of a fluid sample through the channel. The flow rate is linked to the viscosity of the sample, enabling viscosity to be calculated. Furthermore, the flow rate can, in embodiments which require this, be used to calculate the concentration of an analyte measured directly or indirectly by a flow rate dependent detection system. In this case, the flow rate and concentration of the species being detected can all be extracted from the same data signal.

In the present invention, a chamber is provided which contains at least one, and preferably two or more, features which change the cross-sectional area of the chamber. As the flow rate detection means is flow sensitive, these discontinuities can be detected and so the height of sample in the chamber can be known at that point in time. The chamber delivers a volume or volumes of sample to the flow channel such that the volume flow rate of the delivery can be obtained from the recognisable event marker(s) in the measured signal.

In essence, given the known chamber dimensions, detection of the fluid sample passing changes in the profile of the chamber enables flow rate and viscosity of a fluid sample to be calculated. This can then be used to calculate the concentration of an analyte, directly or indirectly, despite any flow rate dependency of the detection means.

If a sample is placed in a chamber above the channel with constant cross sectional area, the height of the sample drops according to:

"- "S" (2, where P is the flow resistance characteristic of the particular channel design.

The volume flow rate, V_f or dV/dt, can be written as AdWdt, from which it can be seen that V_f is related to the change in height by: dH ₌ V_L dt A (3) where A is the cross-sectional area of the reservoir at the top of the solution.

A change in the cross-sectional area from A_r and A₀ is introduced into the chamber at a known height, H_0j so that substituting equation (2) into equation (3) and differentiating gives:

' P (4) When electrodes are placed in the flow channel beneath the chamber such that from equation (1), I₀, at the cross section profile change (ie t=0 in equation (2)) then becomes:

(MH 1 /3

/„ =

(5) At the feature in the internal profile of the chamber, there will be a sudden decrease in I₀ if the value of A decreases. This sudden change in current marks I₀ at H₀ (a "marker") and according to one embodiment of the invention, to obtain the concentration information, the current must be taken at this known and marked point. Alternatively, the variation of current with time may be recorded and used to calculate in retrospect the current at that known and marked point.

Multiple changes in the internal profile of the reservoir would create multiple markers as the sample head passes each change in A at heights H₀, H-, H_π. In other embodiments of the invention, with two profile markers at H₀ and H* in the chamber, the flow rate V_f can be obtained directly from the time between two current discontinuities i.e. the time taken for a constant known volume of solution to drain from the reservoir between H₀ and H*.

Maintaining the convention used above for the solution at an upper profile marker of / = 0, H = H„ and Ai_m ⁼ t_o, when the solution arrives at the lower constriction let t = t H = H and /■^•„, = I\. This will give:

(kAHΛ 1 /3 fkAHΛ 1/ 3

-1. / 3P

\ΓTJ (6) and comparing equations gives

but on substituting for I it becomes

Combining with equation (4) gives an expression for flow rate at all times, t, that the top of the solution in the reservoir is between the constrictions:

This value can be obtained independently of the magnitude of the measured current values, since it depends only on the time between discontinuities in the current trace and not on the values at those discontinuities.

For any channel/chamber unit with n+1 chamber profile features, a calibration factor (Cal = (H,/H,), Cal] = ( //H₂) Cal„ = (H^H„_+t.) can be given which remains associated with the design and dimensions and does not depend on the nature of the sample.

The volume, V, in the reservoir between the constrictions H₀ and H, can be expressed just in terms of: AH V = A(H - H )= —^± (Cal - l) Cal (10) so that V_j can be described for 0<t<t,.

It is also recognised in an alternative embodiment that specific discontinuities in cross-sectional area are not required and that the analytical system could equally function with any chamber conformation which would produce a characteristic alteration of flow rate dependent signal. Specific step changes are not required as it will be clear to one skilled in the art that the passage of sample past alternative continuous chamber profiles can still be established from the result flow rate dependent signal. Deconvolution of the measured flow rate dependent signal allows the time when the sample passed particular locations within the chamber to be established. Indeed, an arbitrary shaped chamber can be seen as being composed of an arbitrarily large number of separate step changes.

In a first example embodiment of the invention there is provided apparatus and a method for measurement of the flow rate of a fluid sample, thereby enabling calculation of its viscosity. Also, the flow rate information could be used in the calculation of the concentration of an analyte in the fluid sample.

The essential elements are a chamber with at least one discontinuity or other change in cross-sectional area and a channel with a flow-rate dependent detection system. The flow-rate dependent detection system may detect reagent present in the sample or added to the sample by some means.

A sample, possibly containing the component to be analysed is placed in a sample chamber 1 having, in this example, two discontinuities in cross-sectional area 2 (giving H₀) and 3 (giving H_jJ. Sample is delivered from the sample to a channel 4 with well-defined flow characteristics and thereafter flows to a sample collection zone 5 which is able to contain the entire volume of the sample when it has drained from a full sample chamber 1 into the channel 4. An optical detection system 6, including source and coupling optics is provided, as is an optical detector 7 such as a photomultiplier tube, photodiode etc. Figure 2 shows a related embodiment in which electrochemical detection is used. In this example, the component to be detected is an electrochemically oxidisable or reducible substance. The electrochemical detector comprises a working electrode 8, e.g. Au, Pt, Ag, C etc., a secondary electrode 9, e.g. Pt, C etc and a reference electrode 10 e.g. Ag/AgCl. The electrodes 8-10 are selected depending on a particular species to be measured. In this example, only one discontinuity, 2, is provided, the location if which gives H₀.

The electrochemical detector is used to estimate (eg chronopotentiometrically or coulometrically) the quantity of said oxidisable or reducible substance. In preferred aspects, the application of the invention includes a fluid flow channel, which contains at least 2 electrodes, each electrode being in electrical contact with the channel. The channel is integrated with a sample supply chamber, which has an internal profile having at least one change in cross sectional area (Figures 2 and 3). The sample is placed in the chamber and it flows from the chamber into the channel. As the sample passes the electrodes, an electrochemical signal is generated which is sensitive to flow rate.

In the examples of Figure 1 and Figure 2, measurements of flow rate can be made. In the example of Figure 1 with two discontinuities, the volume between H₀ and H* gives the volume of the reservoir. The time for this reservoir to drain gives the flow rate V_f. In the example of Figure 2, only one discontinuity is present. In this case, the resulting change in flow rate when the top of the sample reaches this discontinuity is detected and the flow rate can be calculated either from already knowing the flow rate which that sample type would typically have at H₀ or from the time profile of the resulting flow dependent signal, using the fact that, in most embodiments, the height of the sample will decrease exponentially according to Equation 2.

From the flow rate, the viscosity of the sample can be calculated. Alternatively, the flow rate dependent measurement of a chemical species can now be used to calculate the concentration of a chemical species as the flow rate is now known. When concentration measurements are desired, the chemical species may be an analyte in the original sample or which has a concentration linked in some way with the concentration of analyte in the original sample. The concentration of a sample may be obtained from the electrode signal measured between t=0 and t=tι- n additional chamber features could be added to achieve further measurements between say t= t_n_* and t= t_n.

In a further embodiment of the present invention, the chemical species which is measured is produced within the analytical sample at a reaction zone. One example comprises placing the sample, possibly containing component to be analysed, in the chamber from where it is delivered to a reaction zone sensing area and thence to an electrode sensing area in the channel, and allowing it to produce directly or indirectly a corresponding quantity of an electrochemically oxidisable or reducible substance into said channel at the reaction zone and then electrochemically estimating the said oxidisable or reducible substance in said channel at the electrode as a measurand of the quantity of said component to be measured. In one aspect, the said reaction zone comprises a thin layer of reaction component(s), overlaying the channel wall, upstream of the electrode sensing area; the said electrode sensing area comprising at least two electrodes as described also above. In an alternative aspect, the said reaction zone comprises reaction component loaded onto a solid support, and filling across the channel at or near the base of the reservoir. The reaction components include chemical and biochemical reagents eg enzymes, cells, metals, redox reagents.

In an alternative embodiment of the invention the measurement of a component of a liquid system is provided, which comprises placing the sample, possibly containing component to be analysed, in the chamber from where it is delivered to a reaction zone sensing area and thence to an optical sensing area in the channel (figure 1), and allowing it to produce directly or indirectly a corresponding quantity of a substance which exhibits light-absorbing or luminescence and fluorescence properties into said channel at the reaction zone and estimating the said substance in said channel at a detector (eg photodiode) as a measurand of the quantity of said component to be measured. In these embodiments the material from which the channel is formed is transparent and optically coupled to the photodetector and light source (where required) with suitable components (eg prisms, lens, mirrors).

The invention also provides for the use of multiple chambers used consecutively or in tandem with one channel or several channels. In another aspect of the invention, amperometric electrodes may be placed in the flow channel and a method is provided whereby the viscosity of a sample may be measured. The viscosity of the sample will influence both flow rate and diffusion of species to the electrode. The diffusion coefficient, D, which expresses this is a factor of the Levich equation (equation (1)) and is related to the viscosity, η, using the Stokes-Einstein equation:

k_RT

D = ^6π7? (1 1) Where k_B is the Boltzmann constant, T the temperature and a the hydrodynamic radius. Combining equations (1) and (*) to predict the relationship between limiting current and viscosity gives:

Jim k"

(V f ⁾) (n^η))² (12)

where k" will be constant for a particular flow cell, electroactive species and concentration.

Where only flow rate and/or viscosity are to be measured, and no analyte concentration is to be calculated, the magnitude of the signal measured by the flow rate dependent detection means is not of interest; only the time variation of the signal is required for flow and/or viscosity measurement.

The invention also encompasses a computer program to calculate the flow rate, viscosity or analyte concentration of a sample. The computer program takes a time course of readings from the flow rate dependent detection means and uses the above techniques to establish when the fluid sample passed one or more features in the cross-section of the chamber. Using the known dimensions of the chamber, the flow rate or viscosity of the fluid sample can then be calculated. In embodiments where analyte concentration is to be measured, the computer program then use the known flow rate or viscosity to calculate analyte concentration using readings from the flow rate dependent detection means and using the known flow rate / viscosity. The invention also extends to computers adapted to perform the computer program, computer programs stored on or in a carrier, adapted for putting the invention into practice. The program may be in the form of source code, object code, a code intermediate source and object code such as in a partially compiled form, or in any other form suitable for implementation of the processes according to the invention. The carrier may comprise a storage medium, such as a ROM, CD-ROM, semiconductor rom, programmable or field programmable semiconductor, PIC, EEPROM, magnetic recording medium or may be a transmissible carrier such as an electrical or optical signal conveyed via electrical or optical or radio means.

Examples:

The Channel and Chamber: In the example given in figure 1 and figure 2, the channel unit has dimensions of 15mm x 2mm. The depth of the channel is ~0.3mm. These are typical but not exclusive dimensions to achieve laminar flow. Laminar flow is preferred but by no means essential. The channel passes into a collection chamber, which is continuous with the channel, having such volume as able to contain said sample in its entirety (Figure 1 B). According to this example, the channel-chamber unit is limited to one sample, after which it must be emptied or disposed. In an alternative example, figure 3, the outflow is guided through an outflow port 1 1 down a rod 12 (diameter 0.8 mm held in a block in this example) into a lower waste collector 13. According to this example, there is no limit on the number of samples which can pass through the chamber and channel and be collected in the waste. An open-flow channel may also be substituted.

Above the channel is a chamber having as a minimum, one discontinuity in the internal diameter at height H₀ above the channel base, as shown in figure 2. Sample volume measurement is not required, but sample is filled to above the discontinuity. According to the invention the 'discontinuity' in the internal profile of the chamber must cause a change in flow rate as the sample drains from the chamber. Designs causing a change in flow include orifices, nozzles, cones and baffles. In a preferred but not exclusive embodiment (figure 2) this is achieved by a step change in the cross sectional area. In another embodiment, the channel unit has the same dimensions as in figure 2 but the chamber above the channel had two discontinuities at heights H₀ and Hi as shown in figure 1. The volume contained in the chamber between these two heights is a constant given by π(A*/2)²(H₀ - H,), where A, is the cross- sectional area in the chamber between the discontinuities.

The channel and chamber may be oriented in any direction. It will be clear to one skilled in the art that the chamber need not be vertically oriented but may be positioned in any orientation provided that there is some vertical height difference between the sample and the outflow to drive fluid flow.

Further aspects of these embodiments are given whereby the chambers) with the internal profile leading to flow measurement, is produced as a separate unit to the channel (figure 5), the latter containing a detection system. Alternatively, the chamber(s) may be provided integral to the channel, or adapted to be joined to the channel. The invention allows for several chambers to be used together.

Although the channel is shown as horizontal in the accompanying diagrams, it will be recognised by one skilled in the art that it could be positioned in any orientation including being vertical, parallel with the axis of the chamber.

For example, alternative configurations are shown in Figures 12A to 12D. Figure 12A illustrates a chamber and channel where the chamber and channel are vertical and coaxial, with an integrated waste collector and counter electrode functioning as a flow restricting means. Figure 12B illustrates a channel with three step changes in cross-sectional area. Any number of step changes can be provided, indeed, the additional of further step changes allows additional data points to be taken into account to provide a more accurate final measurement. Figure 12C illustrates a chamber having four step changes. Here, the cross section narrows, increases and narrows again twice as fluid drains from the chamber, providing additional data points. Figure 12D illustrates a vertical integrated chamber and channel where individual regions have a curved profile. If the curved profile is calculated so that the remaining sample volume times the cross-sectional area of chamber at the current sample meniscus is constant, the flow rate will remain constant, making measurements and subsequent calculations easier. Figure 5A shows a possible but not exclusive position in a two-step chamber unit for reagents in an analyte sensitive area (eg enzyme, antibody, analytical reagent) which engages in a reaction with a target component of the sample. This chamber unit is 'plugged' into a channel unit during the analytical measurement procedure and liquid added to the chamber to a level above the top discontinuity in the profile, drains into the channel. The chamber unit has a reagent zone sensing area, typically but not exclusively involving immobilised reagent on beads. Figure 5B shows two reagent containing chambers and reagent zone sensing areas 17a, 17b used in serial; the tandem may be plugged into the channel at the start of the procedure or part of the analytical procedure completed before linking to the channel either together or separately. Figure 5C introduces an additional sampling column, plunger or syringe 18, said unit which may also contain reagent to capture or react with a component of the sample to be measured. The walls of the sampling column, plunger or syringe 18 may be modified with capture ligand 19 for the target analyte.

Reagents may also be placed in a sensing area in the channel, upstream of the detector (eg Figure 6) in which there is a sensing area 20 with immobilised reagent at the entry to the Channel and a second sensing area 21 on the same or opposite sides of the Channel. Their position and geometry will affect the resulting flux patterns.

For a particular application, overall flow in the channel may be slowed to extend the residence time of the sample in the unit, if required, with a flow reducer; Figure 4 shows possible but not exclusive position(s) for the reducer. A preferred but not exclusive form for the reducer is a porous plug 14 or 15 at one end of the channel 16 (Figures 4A and 4B); in an alternative design a rod or tube annulus is introduced into the chamber (Figure 4C). In the latter case the tube annulus may also serve to deliver reagents or wash to the channel. A flow reducer may also be implemented by simply having a narrow portion of the channel or using a capillary tube and, in general, the narrowest part of the fluid flow route will limit flow rate.

A detector is placed downstream of the chamber, usually in the channel. In the absence of other 'sensing areas' the detector should give a signal which shows flow sensitivity. Electrochemical measurements are an example of such a measurement. In Figure 2, three electrodes are incorporated in the channel in contact with the fluid flowing through the channel. Electrochemical measurements can be made with this channel. In the presence of 'sensing areas' having a reaction with a component of the sample, with a turnover rate dependent on flow of a compound detected downstream, the detector placed downstream of this sensing area may also give a signal dependent on flow. Simple optical measurements are an example of such a measurement. In figure 1 , the material from which the channel is formed is transparent and optically coupled to the photodetector and light-source.

The detector could also function as the flow rate reducer. For example, an appropriate metal rod could function both as an electrode for an electrochemical sensor and as a flow rate reducer. Similarly, an optical fibre or other optical component could fulfil this function. Where the reaction zone has a solid support, this could also be configured to function as a flow rate reducer.

Particular applications

Measurement of a sample containing an analyte being electrochemically oxidisable:

The core of the flow cell used is described above in figures I -4; the channel can be manufactured as a single or several component system. Typically, but not exclusively it may comprise an open channel or channel unit and coverplate. Overall reduction in flow is achieved with a porous plug. If the channel is to be used for multiple samples, the outflow is guided down a rod (diameter 0.8 mm held in a block) into a lower waste collector (figure 3), else it is contained in a collection chamber which is continuous with the channel (figure 1).

Flow in the channel is characterised using a chamber having one or two discontinuities in the internal diameter as shown in figures 2 and 3. Analysis is performed by filling the chamber with sample. Exact sample volume measurement is not required. Nominal solution height in the chamber is measured from the base of the porous plug.

The channel contains 3 electrodes, each being in contact with the fluid passing through the channel. All potentials are measured relative to a standard Ag/AgCl reference electrode place on one wall of the channel. A working electrode polarised at a potential such that all electroactive species to be measured arriving at it are oxidised or reduced is placed on the opposite wall, together with a counter electrode. In the example here the desired analyte is hydrogen peroxide: hydrogen peroxide is monitored by its oxidation current on a Pt electrode at +600mV vs Ag/AgCl.

In one example a chamber having one discontinuity in its internal profile at height H₀ is used (figure 2). The chamber is filled with sample and begins to drain into the channel, where a current due to the hydrogen peroxide in the sample is measured. t=0 is set as the head of the sample in the chamber passes the step constriction (when H = H„) and the current at this point, I₀, is given by equation 5. If the cross section of the chamber above the constriction is A_r and A₀ at the constriction, then at t=0, H=H₀, there will be a drop in I as the value of A decreases. This sudden change in current marks I₀ at H₀ and hence V_f can be estimated to be able to obtain the concentration information from equation (1), the current must be taken at the point where the sample in the reservoir passes H₀.

In a preferred embodiment, a chamber having two discontinuities in its internal profile at heights H₀ and Hi is adopted. The chamber is filled with sample and the sample drains into the channel where the electrochemical oxidation of hydrogen peroxide is measured. For the cell dimensions here the cell reservoir (volume between the two discontinuities in the chamber) has V= 70μl; the draining of this volume of solution is marked by two features in the current trace produced when the sample meniscus passes the discontinuities; thus V_f can be calculated from the time between the current markers. Thus the current measured from a sample added to this chamber-channel unit can calibrated for concentration: this leads to the curve shown in figure 8.

Determination of an analyte through its conversion to an electrochemically active product due to an immobilised reagent in the chamber or channel.

A reagent loaded column is incorporated placed between the chamber reservoir and channel, said column being typically but not exclusively porous glass. In this example the reagent is an enzyme, glucose oxidase, the analyte is glucose and the product of the enzyme reaction in the presence of its cosubstrate, oxygen, and analyte is hydrogen peroxide. The concentration of glucose is related to the concentration of hydrogen peroxide. The measurement of hydrogen peroxide is achieved by further chemical reaction with a chromogenic or fluorogenic reagents such as luminol, peroxidase/ aminophenazone to produce a species which may be detected optically, in which case the channel illustrated by example in figure 1 is used. Alternatively, it may be determined electrochemically as described in the example above. The chamber-channel unit shown in figure 4A may be used. In this example a porous plug is used to slow the overall flow, placed between the chamber and channel. This is the same chamber-channel unit employed as for hydrogen peroxide determination, together with the incorporation of an enzyme column.

The porous plugs were cut from 2.7 mm thick coarse grade (90 - 150 μm) hydrophilic porous polyethylene, to fit the bottom of the 2mm diameter tube connecting the chamber to the flow channel. The enzyme was loaded onto controlled-pore glass beads of size 125-177 μm which had been amino- silylated by immersion for 2 minutes in a 5 minute-old mixture of 3-aminopropyltriethoxysilane (1ml) in 95 % ethanol (50 ml). In one example, glucose oxidase (GOD) (type VII from aspergillus niger) could be loaded onto the aminated surface using cobalt ions": cobalt (II) chloride hexahydrate (40 mg) and glucose oxidase (20 mg) were dissolved in water (6ml) and amino-silylated glass beads (200 mg) were immediately added. The whole was stirred for 10 minutes then filtered to separate the beads, which were dried in air at room temperature. Exposure to air allowed the cobalt to oxidise to the kinetically inert Cobalt (III). In a second example, poly(glycidyl)methacrylate (PGMA) was precipitated from benzene (1 ml of 10 % solution) onto the aminosilylated glass beads (100 mg) which were suspended in stirred methanol (10 ml). GOD could then be loaded onto the polymer surface"¹: PGMA-coated beads (15 mg) were shaken with GOD (1.5 mg) in borate buffer (1.5 ml of 0.05M sodium borate, pH 8.5) for 24 hours, isolated by filtration, rinsed in water, phosphate buffer, and water again, and dried in air.

Sample, containing the analyte of interest, flows from the chamber into the channel, over the enzyme column where the analyte reacts with the enzyme. In the case of the analyte glucose and of glucose oxidase enzyme reagent, the hydrogen peroxide produced in the enzyme reaction is swept down the channel by the solution flow and is detected at the downstream detector. For the cell dimensions here the cell reservoir (volume between the two discontinuities in the chamber) has V= 70μl the draining of this volume of solution is marked by two features in the current trace produced irrespective of analyte concentration, when the sample meniscus passes the discontinuities; thus V can be calculated from the time between the current markers. An important aspect of the channel flow cell for such a heterogeneous reaction is the well-defined flow of solution and the control over the rate of mass transport. Thus the extent of the enzyme reaction with the glucose substrate in this case is limited by the residence time of the solution within the enzyme-loaded plug and by the amount of enzyme and its cosubstrate, oxygen, which is available to convert the glucose. At low loadings of enzyme, maximum conversion of the analyte is not reached and the amount of hydrogen peroxide measured between the two current markers increases as flow rate decreases. In this condition the chamber-channel unit can be calibrated for glucose, showing an inverse relationship between hydrogen peroxide produced and V_f; this relationship is incorporated into the calibration of the signal measured (eg UV-Vis, Figure 1). In a preferred embodiment when electrochemical detection is employed, high enough amounts of enzyme and low enough flow rates may be used, such that the turnover of analyte reaches a maximum independent of flow rate, limited here by the supply of cosubstrate, oxygen. Data obtained under these conditions can be calibrated for glucose in the same manner as for the example given for hydrogen peroxide determination above. Some typical data is shown in figure 9.

Determination of the 'toxicity' of a sample through its inhibition of an enzyme:

The channel shown in figure 3 was used. 3 electrodes where included in the channel during manufacture: A Pt working electrode, a Pt counter electrode and a Ag/AgCl reference electrode. The working electrode was poised at 0. IV vs Ag/AgCl using a potentiostat to measure the electrochemically oxidisable product of the enzyme reaction. Chronoamperometric data from the flow cell were collected for each sample. The channel-chamber unit was used in high through-put 'batch' mode by refilling the reservoir (~ 200μl) each time it drained below the bottom chamber discontinuity.

In this example a porous plug is used, to slow the overall flow, placed between the chamber and channel. An enzyme loaded column in used as described for the previous example for the determination of glucose. The porous plugs were cut from 2.7 mm thick coarse grade (90 - 150 μm) hydrophilic porous polyethylene, to fit the bottom of the 2mm diameter tube connecting the chamber to the flow channel. The enzyme was loaded onto controlled-pore glass beads of size 125-177 μm which had been amino-silylated by immersion for 2 minutes in a 5 minute-old mixture of 3- aminopropyltriethoxysilane (1ml) in 95 % ethanol (50 ml). The beads were then coated with polymer containing glycidyl groups to allow covalent anchoring of the enzyme: poly(glycidyl)methacrylate (PGMA) was precipitated from benzene (1 ml of 10 % solution) onto the aminosilylated glass beads (100 mg) by slow addition of the benzene solution to a rapidly stirred suspension of the glass beads in methanol (10 ml). Alkaline phosphatase (ALP) could then be loaded onto the polymer surface"": PGMA-coated beads (1 g) were shaken with a solution of ALP (10,000 units, as supplied) in diethanolamine buffer (5 ml of 1 M diethanolamine, 0.5 mM magnesium chloride, 0.1 M potassium chloride, pH 9.8) for 48 hours at 4 °C, isolated by filtration, rinsed in buffer and then distilled water (x3) and dried in air, all at 4 °C. 4mg of enzyme loaded glass beads were placed above the porous plug in the region at the base of the chamber, below the lower discontinuity. The amount of enzyme on a column is kept low (compared with glucose determination method). The enzyme column therefore converts only a small fraction of the substrate passing through it, and so shows the expected simple inverse relationship between concentration and flow rate. The enzyme activity in the plug can thus be determined. Typical but not exclusive potential substrates for this enzyme are the phosphates of naphthol, vanillin, 4-methylaminophenol sulfate, 4-aminophenol, hydroquinone, which produce an electrochemically oxidisable or reducible product of the enzyme reaction.

The presence of an inhibitant in the sample is measured by mixing the said sample with the phosphate substrate for the enzyme and adding ~ 200μL into the chamber. The following estimation of the inhibition of conversion of the phosphate by the enzyme to the electrochemically oxidisable product is obtained from the current measured at the channel electrodes. Two types of information can be obtained by comparing the activity of the enzyme in the absence of inhibitant with the activity of a sample possibly containing inhibitant: the rate of inhibition of the enzyme and the equilibrium turnover of the enzyme. The enzyme activity can be expressed for a column as the turnover rate, T: T = c_a. V_f Thas the units of mole per second when c_α is expressed in moles/dm³ and. V m cm³/s. The flow rate is determined from the time between the two constriction events. Typical data for the inhibition of ALP by vanadate is given in figure 10.

Measuring viscosity of a sample

In this example a channel-chamber unit such as figure 1, figure 3 or figure 4C is used: said chamber having two steps (sequential discontinuities) defining a volume of sample and the draining of this volume of solution is marked by two features in the current trace produced when the sample meniscus passes the discontinuities. Thus V_f can be calculated from the time interval between these current markers and is related to sample viscosity according to equation (12).

According to these examples, overall flow is slowed in one of two ways, either with porous plugs or with an annulus. Plugs were made from 2.7 mm thick coarse grade (90 - 150 μm) hydrophilic porous polyethylene, cut to fit the bottom of the 2 mm diameter tube connecting the chamber to the flow channel. The annulus was constructed by placing a 1.6 mm external diameter rod or tube along the axis of the reservoir and extending it along the length of the 2 mm tube connecting the reservoir to the flow cell. It was maintained in a central position by supports. The overall reduction in flow provided by this constriction is expressed by the formula:

The necessary electrode current signal may be obtained by adding an agent to the sample which may be electrochemically oxidised or reduced. However, in most cases this is unnecessary with aqueous samples containing dissolved oxygen, since the electrochemical reduction of oxygen may be used to provide the marker signal. Concentration is not obtained here from the data (although it could be if required), but the current only used as a marker of flow. The channel required is the same as described above for enzyme inhibition data, except a Au working electrode is employed in this example. Chronoamperometric data from the flow cell is collected at -0.6 V vs Ag/AgCl where oxygen is reduced. The channel-chamber unit was used in high through-put 'batch' mode by refilling the reservoir (~ 200μl) with a new sample each time it drained below the bottom chamber discontinuity.

The sample (solutions of glycerol having viscosity in the range 1 - 20cP) is added to the chamber (~200μL) and the current monitored due to the electrochemical reduction of dissolved oxygen. Discontinuities are observed in the current signal (current markers) as the sample meniscus passes the discontinuities in the cross-sectional area of the chamber. Obtaining flow rate V_f, from the time interval between current markers gives a linear relationship of a wide range of viscosities (see figure 1 1).

Performing a labelled sandwich immunoassay

A typical but not exclusive procedure is as follows as illustrated in Figure 7 A. Chamber la has a reagent column 22 having typically, but not exclusively, immobilised reagent on beads; the reagent being selected to have affinity for the target antigen analyte. In this example, the column 22 was prepared with IgG antibody against an epitope of the target component of the sample. The antibody was loaded onto control led-pore glass beads of size 125-177 μm which had been amino-silylated by immersion for 2 minutes in a 5 minute-old mixture of 3-aminopropyltriethoxysilane (1ml) in 95 % ethanol (50 ml). The methods used are the same as for loading with enzyme as described above. An enzyme labelled antibody against a second epitope of the target component and capable of sandwiching the component complexed with the first antibody is used as a soluble reagent. Chamber lb has a reagent column prepared with an antibody to the labelled antibody 24. Chamber lb may be 'plugged' into the channel at the beginning of the procedure or else before the final measurement step. Chamber la is 'plugged' into Chamber lb. Sample of known volume which might contain target antigen is added to Chamber la and drained to waste. Antigen present in the sample is captured in Chamber 1 a according to the binding equilibrium. Labelled sandwich antibody of known concentration and volume in added to Chamber la and drained through Chamber lb to waste where 25 is reagent zone sensing area 22 after capture of any sample antigen 23 and 26 is a reagent- zone sensing area typically but not exclusively involving immobilised reagent on beads; this reagent has an affinity for the labelled antibody. 27 is zone 26 after capture of any labelled antibody The labelled antigen is captured in Chamber la and Chamber lb relative to the amount of sample antigen that has been bound in the first step in Chamber la. Chamber la is separated from Chamber 1 b, the latter remaining plugged into the channel, and enzyme substrate added ~150μL. The enzyme turnover of substrate is estimated at the electrode in the channel as for the toxicity test above. This will give an estimate of enzyme labelled antibody bound in Chamber lb, which is inversely related to the concentration of antigen in the sample. For a more accurate measurement Chamber la can be plugged into a channel and an estimate of bound sandwich enzyme made by the same method, thus giving a ratio of enzyme label bound in each Chamber.

Figure 7B illustrates a similar procedure for competitive immunoassay in which labelled antigen 28 is added to the sample which may already contain a concentration of antigen 23. The reagent zone 22 captures a mixture of sample antigen 23 and labelled antigen 28, giving zone 29. Residual labelled antigen is captured in reagent zone 30, giving zone 31. Enzyme substrate is added as in the previous example, leading to a measurement of bound labelled antigen from which the concentration of antigen in the original sample can be calculated.

The following documents are referenced herein and are included in their entirety within the present disclosure by way of this reference.

' A. Manz, N. Graber and H. M. Widmer, Miniaturized total chemical analysis systems: a novel concept for chemical sensing, Sensors and Actuators, Bl (1990) 244-248. " T. Laurell and J. Drott, Silicon wafer integrated enzyme reactors, Biosensors and Bioelectronics 10 (1995) 289-299. '" W. L. Benard, H. Kahn. A H. Heuer and M. A. Huff, Thin-film shape-memory alloy activated micropumps, J. Micromech. Sys. 7 (1998) 245-251. ^,v S. Shoji, S. Nakagawa and M. Esashi, Micropump and sample-injector for integrated chemical analyzing systems, Sensors and Actuators, 21 (1990) 189-192. ^v H. T. G. Van Lintel, F. C. M. Van De Pol and S. Bouwstra A piezoelectric micropump based on micromachining of silicon, Sensors and Actuators, 15 (1988) 153-167. " J. Branebjerg, P. Gravesen, J. P. Krog and C. R. Nielsen, Fast mixing by lamination, Proc. IEEE- MEMS '96 Workshop 441 -446. ™ T. S. Lamerink, V. L. Spierling, M. Elwenspoek, H. Fluitman and A. Berg, Modular concept for fluid handling systems, Proc. IEEE-MEMS '96 Workshop 389-394. ^V1" J. J. Gooding and E. A. H. Hall, A fill-and-flow biosensor, Anal. Chem., 70 (1998) 3131-3136 ^1X P. Sosnitza, M. Farooqui, M. Saleemuddin, R. Ulber and T. Scheper, Application of reversible immobilization techniques for biosensors, Anal. Chim. Ada, 386 ( 1998) 197-203. ^x C. E. Hall and E. A. H. Hall, Covalent immobilisation of glucose oxidase on methacrylate copolymers for use in an amperometric glucose sensor, Anal. Chim. Ada, 281 (1993) 645-653. ^X1 C. E. Hall and E. A. H. Hall, Covalent immobilisation of glucose oxidase on methacrylate copolymers for use in an amperometric glucose sensor, Anal. Chim. Ada, 281 (1993) 645-653.

Further modifications and improvements may be made by one skilled in the art within the scope of the invention herein described.

Claims

1. An analytical device for measuring the flow rate or viscosity of a fluid sample, the analytical device comprising a chamber and a flow rate dependent detection means, the chamber functioning to hold the sample but allow it to flow therefrom, the flow rate dependent detection means being sensitive to flow of the sample, characterized in that the chamber has at least one feature which causes a change in sample flow rate when the sample is at a location in the chamber and the flow rate dependent detection means is used to establish when the sample is at said location.

2. The analytical device of Claim 1 further comprising a channel, wherein the chamber allows the sample to flow through the channel and the flow rate dependent detection means is sensitive to the rate of sample flow through the channel.

3. The analytical device of any preceding Claim wherein the sample flows under the influence of gravity.

4. The analytical device of Claim 3 wherein the chamber is vertical.

5. The analytical device of any preceding Claim wherein the feature which causes a change in sample flow rate when the sample is at a location is a change in the cross-sectional area of the chamber.

6. The analytical device of Claim 5 wherein the change in cross-sectional area is a discontinuity in cross-sectional area.

7. The analytical device of Claim 5 or Claim 6 wherein the chamber has two or more changes in cross-sectional area.

8. The analytical device of Claim 7 adapted to calculate the flow rate or viscosity of the fluid sample from the time taken by the sample to move from one feature to a second feature.

9. The analytical device of any preceding Claim wherein the detection means comprises a sensor for a component of the sample.

10. The analytical device of any of Claims 1 to 8 wherein the detection means comprises a sensor for a component added to the sample.

11. The analytical device of any of Claims 1 to 8 wherein the detection means comprises a sensor for matter produced responsive to a component of the sample.

12. The analytical device of any of Claims 2 to 1 1 wherein the detection means comprises an electrochemical sensor.

13. The analytical device of any of Claims 2 to 12 wherein the detection means comprises an optical detection means.

14. The analytical device of any of Claims 2 to 13 having a flow-rate restriction means.

15. The analytical device of Claim 14 wherein the flow rate restriction means is a porous plug.

16. The anal^ytical device of Claim 14 wherein the flow rate restriction means comprises an electrochemical sensor.

17. The analytical device of any of Claims 2 to 16 having a waste collection means.

18. The analytical device of Claim 17 wherein the waste collection means comprises a waste guide means and a waste collector.

19. The analytical device of Claim 17 wherein the waste guide means is a rod.

20. An analytical device for measuring the concentration of an analyte in a fluid sample, the analytical device comprising a chamber and a flow rate dependent detection means, the chamber functioning to hold the sample but allow it to flow therefrom, the flow rate dependent detection means being sensitive to the rate of fluid flow, characterized in that the chamber has at least one feature which causes a change in sample flow rate when the sample is at a location and the detection means is used to establish when the sample is at said location and thereby to calculate the rate of sample flow through the chamber at a given point in time.

21. The analytical device of Claim 20 further comprising a channel, wherein fluid flows from the chamber through the channel and the flow rate dependent detection means is sensitive to the rate of fluid flow through the channel.

22. The analytical device of Claim 20 or Claim 21 wherein the flow rate dependent detection means comprises a sensor for the analyte.

23. The analytical device of any of Claims 20 to 22 wherein the analytical device has a reaction zone which functions to produce chemical species in a concentration which depends on the concentration of the analyte.

24. The analytical device of Claim 23 wherein the reaction zone comprises means to convert the analyte to a chemical species which can be detected by a chemical species sensor.

25. The analytical device of Claim 23 wherein the reaction zone displaces or produces detectable matter in response to the presence of the analyte.

26. The analytical device of Claim 23 wherein the reaction zone removes chemical species from a sample in response to the presence of analyte.

27. The analytical device of any of Claims 23 to 26 wherein the reaction zone comprises a layer of reaction component on a channel wall.

28. The analytical device of any of claims 23 to 27 wherein the reaction zone comprises reaction component immobilised on beads in the channel or chamber.

29. The analytical device of any of claims 23 to 28 wherein the amount of chemical species converted, produced or displaced from the reaction zone is independent of flow rate.

30. The analytical device of any of claims 23 to 29 wherein the reaction zone is selected such that its functionality is altered by the concentration of analyte.

31. The analytical device of any of Claims 23 to 30 wherein the reaction zone comprises an enzyme which catalyses a reaction forming a chemical species, wherein the activity of the enzyme is altered by the presence of the analyte.

32. The analytical device of Claim 31 wherein the analyte is any chemical which has an effect on the reaction zone.

33. The analytical device of any of Claims 23 to 32 wherein the reaction zone comprises antibody to an analyte and the reaction zone carries out immunoassays including sandwich and/or competitive immunoassays.

34. The analytical device of any of Claims 20 to 33 wherein the detection means is an optical detection means.

35. The analytical device of any of Claims 20 to 34 wherein the detection means comprises an electrochemical detection means.

36. The analytical device of any of Claims 21 to 35 wherein the sample flows under the influence of gravity.

37. The analytical device of Claim 36 wherein the chamber is vertical.

38. The analytical device of any of Claims 20 to 37 wherein the feature which causes a change in sample flow rate when the sample is at a known location is a change in the cross-sectional area of the chamber.

39. The analytical device of Claim 38 wherein the chamber comprises at least two changes in cross-sectional area.

40. The analytical device of claim 39 wherein the flow rate or viscosity of the fluid sample is calculated from the time taken by the sample to move from one change to a second change.

41. The analytical device of any of Claims 38 to 40 wherein the change in cross-sectional area is a step change.

42. The analytical device of any of Claims 21 to 41 having a flow-rate restriction means.

43. The analytical device of Claim 42 wherein the flow rate restriction means is a porous plug.

44. The analytical device of Claim 42 wherein the flow rate restriction means is an electrochemical sensor.

45. The analytical device of any of Claims 20 to 44 having a waste collection means.

46. The analytical device of Claim 45 wherein the waste collection means comprises a waste guide means and a waste collector.

47. The analytical device of Claim 46 wherein the waste guide means is a rod.

48. The analytical device of any of Claims 20 to 47 comprising a plurality of chambers.

49. The analytical device of any of Claims 20 to 49 wherein a or each chamber has a plurality of detection means for different analytes.

50. A method for measuring the flow rate or viscosity of a fluid sample, comprising the steps of:

(a) adding said fluid sample to a chamber, the chamber being adapted to allow the fluid sample to flow therefrom and comprising at least one change in cross-sectional area, (b) using a flow rate dependent detection means to make a reading sensitive to the sample flow rate, (c) using the detection means to establish when the sample is at one or more known locations with reference to the at least one change in cross-sectional area, (d) thereby calculating the flow rate and/or viscosity of the sample.

51. The method of Claim 50 wherein the fluid sample flows from the chamber into a channel and the flow rate dependent detection means is sensitive to the flow rate of the sample through the channel.

52. The method of Claim 51 wherein the sample flows under the influence of gravity.

53. The method of Claim 52 wherein the chamber is vertical.

54. The method of any of Claims 50 to 53 wherein the change in cross-sectional area is a step change in the cross-sectional area of the chamber.

55. The method of any of Claims 50 to 54 wherein the chamber comprises at least two changes in cross-sectional area.

56. The method of any of Claims 50 to 55 wherein the flow rate or viscosity of the fluid sample is calculated from the time taken by the sample to move from one change to a second change.

57. The method of any of Claims 50 to 56 wherein the detection means comprises a sensor for a component of the sample.

58. The method of any of Claims 50 to 56 wherein the detection means comprises a sensor for a component added to the sample.

59. The method of any of Claims 50 to 56 wherein the detection means comprises a sensor for matter produced responsive to a component of the sample.

60. The method of any of Claims 50 to 59 wherein the detection means comprises an electrochemical detection means.

61. The method of any of Claims 50 to 60 wherein the means comprises an optical detection means.

62. The method of any of Claims 51 to 61 wherein the channel has a flow-rate restriction means.

63. The method of Claims 62 wherein the flow rate restriction means is a porous plug.

64. The method of any of Claims 50 to 63 wherein the fluid flows into a waste collection means.

65. The method of Claim 64 wherein the waste collection means comprises a waste guide means and a waste collector.

66. The method of Claim 65 wherein the waste guide means comprises a rod.

67. A method for measuring the concentration of an analyte in a fluid sample, comprising the steps of:

68. The method of Claim 67 wherein fluid flows from the chamber into a channel, wherein the flow rate dependent detection means is sensitive to the rate of fluid flow within the channel.

69. The method of Claim 67 or Claim 68 wherein the flow rate dependent detection means comprises a sensor for the analyte.

70. The method of any of Claims 67 to 69 wherein the analyte is converted to a chemical species which can be detected by the a sensor for said chemical species.

71. The method of any of Claims 67 to 69 comprising the step of causing detectable matter to enter or leave the fluid sample in a concentration which depends on the concentration of the analyte.

72. The method of Claim 71 wherein a chemical species is displaced or produced in response to the presence of the analyte.

73. The method of any of Claims 71 or Claim 72 wherein detectable matter enters or leaves the fluid sample in a concentration which depends on the concentration of the analyte as a result of the action of a layer of reaction component on a channel wall within the channel or chamber.

74. The method of Claim 71 or Claim 72 wherein detectable matter enters or leaves the fluid sample in a concentration which depends on the concentration of the analyte as the result of the action of a component immobilised on a support within the channel or chamber.

75. The method of any of Claims 71 to 74 wherein the amount of detectable matter which enters or leaves the fluid sample depending on the concentration of the analyte is independent of flow rate.

76. The method of any of Claims 71 to 75 wherein the amount of detectable matter which enters or leaves the fluid sample depending on the concentration of the analyte is responsive to the concentration of analyte.

77. The method of any of Claims 71 to 68 wherein the amount of detectable matter which enters or leaves the fluid sample depending on the concentration of the analyte is controlled by an enzyme which catalyses a reaction forming a chemical species, wherein the activity of the enzyme is altered by the presence of the analyte.

78. The method of any of Claims 71 to 77 wherein the analyte is any chemical which causes detectable matter to enter or leave the fluid sample.

79. The method of any of Claims 71 to 78 comprising the step of carrying out immunoassays, including sandwich or competitive immunoassays, using an antibody to the analyte.

80. The method of any of Claims 67 to 79 wherein the detection means comprises an optical detection means.

81. The method of any of Claims 67 to 80 wherein the detection means comprises an electrochemical detection means.

82. The method of any of Claims 67 to 81 wherein the sample flows under the influence of gravity.

83. The method of Claim 82 wherein the chamber is vertical.

84. The method of any of Claims 67 to 83 wherein the change in cross-sectional area is a step change in cross-sectional area.

85. The method of any of Claims 67 to 84 wherein the chamber has two or more changes in cross- sectional area.

86. The method of Claim 85 comprising the step of calculating the flow rate or viscosity of the fluid sample from the time taken by the sample to move from one change to a second change.

87. The method of any of Claims 67 to 86 wherein the channel has a flow-rate restriction means.

88. The method of any of Claims 67 to 87 wherein the flow rate restriction means is a porous plug.

89. The analytical device of any of Claims 67 to 88 wherein the flow rate restriction means comprises an electrochemical sensor.

90. The method of any of Claims 67 to 89 wherein the fluid sample flows into a waste collection means.

91. The method of Claim 90 wherein the waste collection comprises a waste guide means and a waste collector.

92. The method of Claim 91 wherein the waste guide means is a rod.

93. The method of any of Claims 67 to 92 comprising the step of using multiple chambers.

94. The method of any of Claims 67 to 93 comprising the step of using more than one detection means for different analytes per sample.

95. A chamber for holding a fluid sample and allow said fluid sample to drain under the influence of gravity, the chamber comprising at least one change in cross-sectional area.

96. The chamber of Claim 95 wherein a change in cross-sectional area is a step change in cross- sectional area.

97. The chamber of Claim 95 or Claim 96 comprising at least two changes in cross-sectional area.

98. A computer program for determining the flow rate or viscosity of a fluid sample inserted into and flowing from a chamber, the computer program comprising program instructions for

a) analysing a time course of readings from a flow rate dependent detection means to determine when a fluid sample passed one or more features in the cross-section of the chamber; and

99. The computer program of Claim 98 wherein the features are step changes in the cross- sectional area of the chamber.

100. The computer program of Claim 98 or Claim 99 wherein the flow rate or viscosity is calculated from the time taken for the sample to pass between two or more features.

101. A computer program for determining the concentration of an analyte in a fluid sample inserted into and flowing from a chamber, the computer program comprising program instructions for

c) thereby calculating the analyte concentration.

102. The computer program of Claim 101 wherein the features are step changes in the cross- sectional area of the chamber.

103. The computer program of Claim 101 or Claim 102 wherein the flow rate or viscosity is calculated from the time taken for the sample to pass between two or more features.