GB2478225A - Monitoring the density or viscosity of a sample using a multi-beam resonator - Google Patents

Abstract

A device for monitoring a change in the density and/or viscosity of a test sample using a multi-beam resonator comprising; a reaction chamber defining a volume for receiving and retaining a test sample; at least two resonant beam members 302, 303 within the reaction chamber; at least one vibratory element 307 positioned to cause the vibration of at least one of the resonant beam members; and at least one sensor means 307 for determining at least one parameter associated with the vibration of the resonant beam member. A mounting zone (d, Fig 6) of the multi-beam resonator is provided and the ratio of the total longitudinal length of the resonant beam members to the length of the mounting zone is in the range 1 to 9. The vibratory element and sensor means may both be piezoelectric devices and the device may form part of a test strip or test kit.

Description

I

APPARATUS AND METHOD FOR DETERMMNG THE RESULTS OF

ASSAYS

R&d of the kivention The present invention provides an improved sensor apparatus for the measurement of assays and chemica reactions wherein a change in viscosity and/or density, by for exampe, geVation, precipitation, agg'utination or coaguation, occurs. n particuar, there is provided a biosensor for use in the rea time measurement of an assay, such as an endotoxin assay used to detem,ine the presence of microbia contaminants in a test sampe, the apparatus aflowing the viscosity and/or density of the sampe to be measured continuousy as the assay progresses. The invention further extends to methods for performing such assays, and further to the use of said apparatus in the performance of such methods.

Background to the hvention

The viscosity of a fluid may be measured using a viscometer. Viscometers measure the properties of a fluid under conditions where the fluid remains stationary and an object, such as a vibratory eement moves through it.

The measurement of the viscosity can be determined based on the drag caused by the r&ative motion of the fluid and surface.

One specific type of viscometer is a vibration viscometer. Vibration viscometers typically function by allowing the measurement of the dampening of an oscillating &ectrochemica resonator which is immersed in the fluid of which the viscosity is to be determined. The greater the viscosity of the fluid, the arger the dampening effect imposed on the resonator.

A change in the density or viscosity of a fluid can accompany or be the resut of the occurrence of a chemica reaction. A change in the density or viscosity of a test sample can, in particuar, occur during the performance of assays commonly used in areas such as haematology where blood dotting, or coagulation reactions are measured, in immunology where immunoassays are performed, and further for more general screening, such as high throughput screening, for example to detect the presence of a contaminant or analyte in a sample.

One example of such an assay which uses a change in density or viscosity of a test sample in order to provide result is assay based test to determine the presence of bacterial endotoxin in a test sample.

Bacterial endotoxin, such as lipopolysaccaharide (LPS), is a fever producing product of gram negative bacteria. Endotoxins typically have a hydrophobic core which is bound to repeating ollgosaccharide side chains.

When present in the bloodstream, even in low levels, endotoxins can cause fever, shock, hypotension, raised erythrocyte count, and disseminated intravascular coagulation. If the endotoxin is present in a sufficient high level in the bloodstream, it can cause death.

The US Food and Drug Administration department (FDA) requires drugs and pharmaceutical compounds which are to be administered to a subject either by injection, or intravenously, to undergo an endotoxin test prior to their administration. Furthermore, prosthetic devices, such as heart valves or hip replacements also require such an endotoxin test. There is therefore an ongoing need to provide accurate testing to determine the presence of endotoxin within a test sample.

The traditional test used to identify the presence of pyrogens, such as endotoxin, was the rabbit pyrogen test. This test, which dates back to 1942, suffered the disadvantages of being both slow and expensive to perform. Subsequent to this, Levin and Bang discovered that blood ceDs (amebocytes) which were derived from the horseshoe crab (Limulus polyphemus), contained a dotting agent that attaches to the endotoxins produced by gramnegative bacteria, This clotting agent was identified as Limu/us amoebocyte ysates, wNch is commonly abbreviated to "LAL.

LAL was used to develop a number of geHation based dotting tests which detect and quantify the presence of endotoxin within a test sample, A number of such LAL-based tests are currently used commerciafly to determine the presence and quantity of endotoxin within a sample.

For example, the gel-dot test is a sample test that uses a lysate preparation of Limulus polyphemus blood to give a positive / negative answer by means of the presence or absence of gel-clot formation within a test tube or viaL The presence of endotoxin results in dotting, and hence, this change of state can be visuay determined by inverting the vessel in which the reaction has occurred.

Preparations of LAL lysate are commerciaHy available in different sensitivities, to give a qualitative. In addition, testing kits that contain LAL reagents may be provided in ready to use' tube vials, which allow for dfluted samp'es to be added to the vials so that the assay can be performed within them.

Gel-dot kits are frequently used for field testing. They may also be used in laboratories where small sample throughput is required. The maximum sensitivity of the gel-clot test is generally a level of around 0.03 EU/mI of endotoxin in a sample. However, there are disadvantages associated with such tests, in particular the cost as a large quantity of LAL reagent must be used for each sample. Furthermore, the results from the gelclot LAL test are subject to human interpretation, as the results are visuaHy determined. An assay based on the subjective determination of a gel-dot is particularly difficult to automate, hence determining the result of the gek dot test in this way may introduce a greater degree of error into determining the results of the test, and may further lead to the increased risk of reporting a false negative result.

A further technique used to identify endotoxin in a sample is by means of optical analysis: chromogenic or turbidometric methods. These methods can be fufly automated, and further have the advantage of providing quantitative results. These tests can also use a "kinetic" approach, measuring a response as a function of time can be obtained, rather than waiting for the entire reaction to complete before determining the result.

The turbidometric method is a sensitive LALbased method used for determining endotoxin levels in a sample, with a maximum sensitivity of 0.001 EU/mI. However, for this level of sensitivity the methodology requires the use of costly, large and sensitive laboratory equipment, and therefore analysis of this type is restricted. Equipment which allows for "kinetic" LAL testing to be performed on a smaller scale, albeit with lower sensitivity is commercially available; however such systems (for example ENDOSAFETM, Charles River Laboratories) use microfluidics and optical technology to analyse the sample within a tiny sampling area. Such approaches have limited sensitivity and poor precision due to the short pathlength of the interrogating light through the sample. The resulting method will be suffer increases in analysis time and decreases in precision with a desirab'e reduction in sample voume that enable reductions in reagent and samp'e usage A quartz crysta' microbalance (QCM) may be used for detecting the end point of a geflation or aggutination reaction. For example a LAL assay can be performed by immersing the crystal within a test sample undergoing a LAL reaction. A key failing of resonating crystals is caused due to the high frequency of resonance of a pure piezoelectric resonator (for example in European Patent No 0,304283 Bi these values are in the order of 6 12MHz). The depth of the liquid probed by the vibration wifi therefore be very smafi, and so it is to be expected that the signal will also be very smafi. However, WO 2005/114138 teaches that applying a texture to the surface of a planar resonating device operating at a megahertz frequency improves the viscosity and density measuring capability. The textured surface creates an entrapped layer of liquid close to the sensor surface.

In the case of monitoring of biological reactions, this not ideal because the sensor now responds according to the kinetics of the diffusion of the reagents and reaction products in the entrapped layer, rather than responding to the reaction in the bulk. In order to address these shortcomings in the devices known in the prior art, the inventors have identified that ideafly a lower frequency resonator, for example of 400 kHz or less, would be optimal for the measurement of biological reactions.

US 6,401,519 teaches how mechanical oscillators can be used to analyse the properties of fluids. Different viscosities and density values present different characteristic frequency/amplitude responses. A twin beam cantilevered tuning fork as shown in US 6,401,519 suffers from energy losses due to moments acting at the mountings of the beams. The clamping/mounting of electrodes or coatings at the mountings of the beams will create wide variations in sensor performance. The highloss twin beam cantflever tuning fork has Utile energy to resonate in Uquid and is a'most competSy damped. Furthermore, the twin cantflevered arrangement disdosed cannot be reUaby waterproofed as coating the mountings cause this to be damp the signas further.

Following extensive experimentation, the present inventors have surprisingy provided an improved apparatus and method for use in performing rea time monitoring of the progress of an assay or chemica' reaction, wherein the assays or chemica reaction resufts in a change or density or viscosity of a test samp'e, typically by way of the production of a get, precipitate, coaguate, precipitate or the Uke. n particu'ar, the inventors have provided an apparatus and method for the detection of endotoxin in a test sampe using amoebocyte ysates, wherein the progress of the gellation of the test sampe can be monitored in rea time.

The methods of the invention are advantageous in as far as they provide a quantitative assay resuft, and as such, when empbyed in r&ation to assays, such as the determination of bacteria endotoxin contamination in a sampe, they provide the further associated advantages of an enhancement in sensitivity, specificity and throughput, when compared to LALbased endotoxin tests currenily known in the art.

Summary of the kwention

According to a first aspect of the invention there is provided a method for the monitoring, in reakime, of a change in the viscosity and/or density of a test samp'e which is undergoing a chemica reaction, the method comprising the steps of: providing a fluid test sample, optionally adding at Heast one reagent to the test samp'e to form a test sample mixture, immersing at least one resonant beam member of a multi-beam resonator comprising at least 3 resonating beam members in the test sample mixture, and determining a change in viscosity and/or density of the test sample mixture by monitoring the change in at least one parameter relating to the resonance of at least one of the resonating beam members, said parameter being selected from the group consisting of; resonance frequency, quality factor and variation of the resonance phase angle of said at least one resonating beam member, and using the change in said at least one parameter to calculate the viscosity and/or density of the test sample mixture in order to determine the occurrence of a chemical reaction within the test sample mixture.

In certain embodiments, the occurrence of a chemical reaction within the test sample mixture is indicative of the presence of an analyte, contaminant, antigen or antibody within the test sample mixture. The observed increase in density and/or viscosity of the test sample mixture may be due to the occurrence of the chemical reaction resulting in at least one of a gelation, agglutination, coagulation or precipitation occurring within the test sample mixture.

In one embodiment, the step of determining the change in the viscosity and/or density of the test sample mixture by means of monitoring a change in at least one parameter selected from the group consisting of; resonance frequency, quality factor and variation of the resonance phase angle of the immersed resonant beam member(s), is performed by defining said at least one parameter relative to the same parameter as derived from at least one further resonant beam member of the triple beam resonator, said at least one further beam member also being immersed within the test samp'e mixture. in certain embodiments, the change in the parameter may be monitored continuou&y from a time point when the resonant beam member(s) is immersed in the test sampe, or from when the reagent is added to the test sampe to form the test sampe mixture. Monitoring the parameter in this way aVows the continuous, reap time monitoring of the progress of any chemical reaction which may be occurring in the test sampe mixture, which resufts in a change in the density and/or viscosity of the sample.

in certain embodiments, the data obtained foVowing the anaysis of the at east one parameter, such as Q4actor, determined in re'ation to the physica resonance properties of the resonating beam members of the test strip may be processed using at east one agorithm which can be used to process this input data and determine whether a change in density and/or viscosity of the test sample is occurring. In certain embodiments, the algorithm wUl be stored by, and used in the calculation of output data, by an electronic processing apparatus, such as a computer.

In certain embodiments, the output parameter data relating to the at least one resonating beam member is communicated to the processing apparatus as at least one signal, which is in turn communicable to a user to indicate the presence or absence of bacterial endotoxin in the sample, due to an increase in viscosity due to gellation of test sample.

Typically, the test sample is a fluid test sample, in particular a liquid. Said liquid may be selected from the group consisting of, but not limited to: a biological sample, such as a pharmaceutical composition or a liquid for use in the same; a bodily fluid, such as blood, a blood product, or the like; or any other liquid test sample which is to be analysed for the presence of a parameter, such as a contaminant, an analyte, an antigen, an antibody or the fike, wherein a change of density and/or viscosity may resut in the samp'e during the testing process.

As herein defined, the term 4quaty factor' means a unit used to measure the qua'ity of a resonant system; specificaHy it is a measurement of the sharpness of resonance or frequency seecUvity of a resonant vibratory system. n aD resonating devices, the quaUty factor is affected by the surrounding environment. The qu&ity factor of a resonant system changes according to the viscosity of the media in which it osciDates.

Accordingy, as the fluid becomes more viscous, this resu'ting in an associated change in the quahty factor of the fundamenta' resonance of the resonating beam structures described herein. The determination of the quahty factor (Q4actor) typicafly aVows the rate of change of the viscosity of the samp'e mixture to be determined.

As herein defined, the terms "resonance phase angl& re'ates to a measurement of the difference in phase of the resonance of one resonating beam member reative to the phase of at east one further resonating beam member, said second beam member being paraDe to the first resonating beam member.

As herein defined, the term "resonance frequency" means the frequency when a materia, in this case a resonating beam member of a mutibeam resonator, resonates at maximum ampUtude at a specific mode of resonance. The frequency of the resonating beam wiD change reative to the viscosity of the fluid in which it is immersed. The invention therefore provides a mutibeam resonator viscometer device which comprises a puraflty of resonating beam members, where the frequency of at east one of the vibrationa' resonant beam members aVows a change in density and/or viscosity of a fluid in which it is immersed to be determined. The frequency of the resonating beam member as detected may not &ways be the resonant frequency.

n certain embodiments, the determination of the change in density and/or viscosity of the test sanipe mixture is performed continuous'y by monitoring the density and/or viscosity of the test sampe mixture. Such continuous monitoring may be referred to as dynamic monitoring of the density or viscosity of the sampe mixture. According!y, in certain embodiments, the method of this aspect of the invention aVows the rate of change of density and/or viscosity of a sampe mixture to be measured.

n certain embodiments, the determination of the change in density and/or viscosity of the sampe mixture may be determined by the taking of a series of readings to determine the parameter of at east one of resonance frequency, resonance phase ange and/or quafity factor, said readings taken at specific time points, wherein the data obtained from said readings can be ana'ysed in order to determine, for exampe by cacthation, any resulting change in the density and/or viscosity of the sampe mixture. n certain embodiments a reading is taken every 80 seconds during the progress of the chemica reaction within the sampe. n certain further embodiments, the frequency of this reading can be any vaued from under I second to over 1 minute.

n certain embodiments, the data reating to at east one parameter which is derived from the resonance of the resonating beam member may be processed, for exampe, using an agorithm, in order to determine whether there has been a change in the density and/or viscosity of the samp'e mixture. Accordingy, in various further aspects, the invention extends to an agodthm, and further to the use of such an agohthm for processing data r&ating to the parameters identified hereinbefore as dehvabe from the resonating beam members in order to provide a numerica' vakie which can be used in the determination of the density and/or viscosity of the test samp'e mixture.

n certain embodiments, the data re'ating to the at east one parameter which is determined from the at east one resonating beam member can be compared to known vaues in order to determine whether there has been an associated increase or decrease in the density and/or viscosity of the sampe mixture.

n certain embodiments, an increase or decrease in the density and/or viscosity of the sampe mixture can be determined by detecting a change in the resonating frequency of at east one resonating beam member of the mutibeam resonator, typicafly a reduction in frequency for an increase in density. n gener&, the determination of resonant frequency is a more direct function of density than quaHty factor (Q$actor) which is more reated to the viscosity of the fluid.

n certain embodiments, an increase or decrease in the density and/or viscosity of the sampe mixture can be determined by detecting a variation in the quaty factor (Q factor), typicay a decrease in the quaity factor for an increase in viscosity. n certain embodiments, a measurement of the rate of change of Q factor is used to determine the rate of change of viscosity of the sampe mixture.

n certain embodiments, an increase or decrease in the density and/or viscosity of the sampe mixture can be determined by detecting an akeration in the resonance phase angie of at east one resonant beam member reative to at east one further resonant beam member.

Typically a change in the density and/or viscosity of the samp'e mixture indicates that the sampe mixture is undergoing gellation, agglutination, precipitation, coagulation or the like. This change is typically caused by the reaction of the test sample with the reagent present in the sample mixture.

In certain further embodiments, the method may further comprise a step or steps to allow the resonant beam members to be calibrated, Typically calibration is performed by way of resonating the resonant beam members prior to immersion in the test sample, in a fluid which has a known density and/or viscosity, most typically air. In certain embodiments, this initial calibration step is performed prior to the test sample mixture immersing the at least one resonant beam member. In certain embodiments, this calibration includes the determination of the temperature in the environment of the multibeam resonator. The determination of the environmental temperature can be important as this may have an effect on the speed of progression of a chemical reaction, such as a coagulation or gellation reaction. As such, in one particular embodiment, the method of this aspect of the invention further comprises the step of calibrating the multibeam resonator prior to immersion in the test sample mixture, the calibration step comprising resonating the resonating beams in air. In certain further embodiments, the calibration step includes determining the temperature by means of determining the inner temperature within an apparatus which may house the multibeam resonator using a thermistor or the like, and further determining the outer temperature of the environment using the multibeam resonator, in order to establish the air temperature which the test sample will be exposed during the chemical reaction which is to be monitored by the resonant beam members of the multibeam resonator, In certain embodiments, the method of this aspect of the invention is an automated method, which aVows for the automatic or continuous performance of the assay method.

In one embodiment, the method may be used to determine the presence and/or quantity of endotoxin which is present within a test sample, wherein this determination is based on the formation of gellation within a test sample.

Accordingly, in such an embodiment the foregoing method would provide a method for the continuous monitoring of a change in the viscosity and/or density of a test sample to determine the presence and/or quantity of bacterial endotoxin within the sample, the method comprising the steps of: -providing a test sample, -admixing the test sample with a reagent comprising amebocyte lysate or a synthetic analogue thereof in order to form a test sample mixture? immersing at least one resonant beam member of a multi-beam resonator device according to the invention in the test sample mixture, and -determining a change in at least one parameter associated with the resonance of at least one resonating beam member selected from the group consisting of: (i) the resonance frequency of the at least one resonant beam member, (ii) the quality factor of the at least one resonant beam member? (iii) changes in the resonance phase angle of the at least one resonant beam member relative to at least one further resonant beam, and -using the observed change in said at least one parameter to calculate the viscosity and/or density of the test sample mixture, wherein an increase in the viscosity and/or density of the test sample mixture is indicative of the presence of endotoxin within the test sample.

In certain embodiments, the determination of the values of the identified parameters associated with the resonance of a resonant beam member within the test sample mixture are performed repeatedly and, typically continuously from the time when the reagent is added to the test sample to form the test sample mixture, or from the time point from when the resonant beam member is immersed in the test sample mixture.

Monitoring the resonance of the resonant beam member in this way allows the continuous, real-time monitoring of changes of density and/or viscosity in the test sample mixture. Tracking the progress of any chemical reaction within the test sample mixture in this way is advantageous in that the method allows for the early identification of the occurrence of gellation, which can be determined by way of an increase in viscosity of the test sample mixture, in order to provide an indication that endotoxin is present within the test sample mixture.

In certain embodiments, the data obtained following the analysis of the at least one parameter, such as Q-factor, determined in relation to the physical resonance properties of the resonating beam members of the test strip may be processed using at least one algorithm which can be used to process this input data and determine whether a change in density and/or viscosity of the test sample is occurring, and whether, therefore, endotoxin is likely to be present in the test sample.

In embodiments where an &gorithm is used to interpret the parameter data, this data may include for example, data relating to a parameter associated with: the frequency of oscillation of at least one of the beam members, the quaUty factor and/or resonance phase angle. Said algorithm can be used to provide a numerical value, which itself, or by reference to a standard derivative, can be used to determine an end point of a chemical reaction, for example of a Lirnulus amoebocyte lysate (LAL) based endotoxin detection screening assay reaction, and further, which can be used to provide a quantitative measure as to whether bacterial endotoxin is present or absent from a specific test sample.

In certain embodiments, the output parameter data relating to the at least one resonating beam member is communicated to the processing apparatus as at least one signal, which is in turn communicable to a user to indicate the presence or absence of bacterial endotoxin in the sample, due to an increase in viscosity due to the geflation of test sample.

In certain embodiments, the output signal may be a visual indication which indicates the presence or absence of bacterial endotoxin in the test sample, and which may further provide a determination of the amount of bacterial endotoxin in the test sample. The indication may be a visual signal such a coloured Ughting or a text or symbol based display, or an aural signal, such as a sound.

As herein defined, the term sendotoxin5 refers to potentially toxic, naturally derived compounds which are derived from pathogenic organisms, in particular bacteria. The endotoxin is therefore typically a structural component of a bacteria which is released when the bacteria is lysed.

Typical examples of endotoxins include lipopolysaccaharide (LPS) and flpooHgosaccharide (LOS) both of which are found in the outer membrane of gram-negative bacteria.

n certain embodiments, the at east one reagent which is mixed with the test sampe is typicafly amebocyte ysate, or a synthetic anabgue thereof.

n certain embodiments, the amebocyte ysate is provided in dried form, with the ysates and is reconstituted during the reaction process which occurs during the screening methods of the invention.

In certain embodiments, the amebocyte ysate may be formuiated with saks and/or buffer in order to stabilise the amebocyte ysates.

Performance of LAL-based endotoxin screen assays is based upon obtaining Limuus amebocyte lysates (LAL) direcily from the horseshoe crab. There are four known species of the horseshoe crab, these being: Limulus polyphemus, Tech ypleus giges, Tech ypleus tridentatus, and Carcinoscoypius rntuhicauda. Limulus amoebocyte lysates (LAL) is obtained by bleeding the crab and deriving the LAL from the obtained blood product. Although this procedure is rarely leads to morbidity for the crab, the associated costs of production for obtaining commerciafly viable quantities of LAL is high. Accordingly, alternative compounds are being developed which wifi replace LAL in endotoxin screening assays. Such compounds, such as synthetic analogues of LAL are within the scope of the present invention.

TAL (Tachypleus amoebocyte lysate) functions similarly to LAL in aiding the detection of gram-negative bacteria. As such, in certain embodiments of the present invention, Limulus amoebocyte lysates (LAL) may be replaced with TAL (Tachypleus amoebocyte lysate) or a synthetic analogue thereof.

Furthermore, the production of a protein by recombinant means is a technique which is widely used in the field of molecular biology. Ongoing work is being performed to identify the gene which encodes for Limulus amoebocyte Dysates (LAL). Identification of this gene, will aVow it to be cloned and expressed by recombinant means, for example, using prokaryotic or eukaryotic cells. A recombinant or synthetic form of Limulus amoebocyte Jysates (LAL) or TAL (Tachypleus amoebocyte lysate) may therefore be provided. Accordingly, in certain embodiments, the invention extends to the use of a synthetic or recombinantlyderived form of Limulus amoebocyte lysates (LAL) or TAL (Tachypleus amoebocyte lysate) which may be used to replace, or to supplement the use of naturally derived Limulus amoebocyte lysates (LAL) or TAL (Tachypleus amoebocyte lysate) in the methods of the present invention.

In certain embodiments, the test sample can be selected from the group comprising of, but not limited to: a pharmaceutical composition, a biological composition or fluid, a parental preparation, such as a diluent, carrier or adjuvant for the preparation of, or for administration along with a biological or pharmaceutical composition, a reconstitution buffer or salt solution for a pharmaceutical composition or the like, an injectable pharmaceutical. In certain embodiments, the sample is a water sample, for example, sterile water, natural water, purified water, treated water or distilled water, where it is required to determine whether said water sample is free from endotoxin contamination.

The method of this aspect of the invention can also be used to follow any other assay or chemical reaction where a gel, solid, precipitate, agglutinate or coagulate is formed in a test sample as a result of a chemical reaction therein.

Accordingly, in one embodiment of the assay of the first aspect of the invention, the assay is an immunoassay, such as an enzyme linked immunosorbant assay (ELISA) which is used to detect the presence of at least one target analyte, such as an antibody or an antigen, in a test sample. In such an embodiment, the reaction between an enzyme labeHed probe and a substrate can generate a precipitate. In such instances, the reagent which is added to the test sample can be an enzymatic substrate which serves to indicate whether the particular analyte is present in the test sample.

In embodiments of the invention, where the chemical reaction relates to an ELISA assay, the reagent may be Tetramethylbenzidine (TMB) (3, 3,5,5'Tetramethythenzidine) or any precipitate forming reagent, the TMB example is used for detecting horseradish peroxidase (HRP) labefled probes, such as antibodies used in an ELISA assay. This provides a convenient nonoptical technique for detecting probes used in optical assays.

In a yet further embodiment of the method of the first aspect of the invention, the method may be used to monitor the progression of a latex agglutination assay where, for example, antibodies are presented on the surface of latex beads, in order to perform an antibody agglutination test.

Such a test can be used to detect the presence of antigens in a reagent system. In such an embodiment, the agglutination of the antibody bound beads indicates the en&point of the assay. During the agglutination reaction, changes in the density and/or viscosity of the test sample about the resonating beams can be detected.

In a yet further embodiment, the method of this aspect of the invention can be used to detect the result of the Widal test, a presumptive serological test for Enteric fever or Undulant fever. The Widal test is widely used in developing countries to test for typhoid fever. The test demonstrates the presence of somatic (0) and flagellar (H) agglutinins to Salmonella typhi in the patients serum using suspensions of 0 and H antigens. Typically the Widal test is performed by the tube agglutination technique. The method of the present invention could therefore be used to monitor the occurrence of agglutination in order to accurately provide a result, In still yet further embodiments, the method of the present invention can be used to monitor the progress of other reactions which involve a chemical reaction wherein a gellation, agglutination, precipitation or a coagulation occurs within the sample mixture, or wherein a partial change of state of the sample mixture occurs, for example, during the selling of a adhesive or resin, such as an epoxy resin, wherein a first part (i.e. a reagent) is added to a second part (being equivalent to what is defined herein as a test sample) in order to monitor the selling of the composition.

In various further aspects the invention provides a multibeam resonator which may be used to perform the methods of the invention.

Accordingly, a yet further aspect of the present invention provides a multi-beam resonator for use in monitoring a change in density and/or viscosity in a test sample, comprising: at least 3 resonant beam members, said beam members being arranged in a substantially parallel arrangement, -at least one vibratory element positioned to cause the vibration of at least one of the beam members, at least one sensor means for determining at least one parameter associated with the vibration of the resonant beam member, -a reaction chamber defining a static volume, which is suitable for receiving and retaining a test sample, the reaction chamber comprising at least one inlet port to allow the introduction of the test sample and at least one outlet port of dimensions which allow for the outflow of air but not the test sample from the reaction chamber, wherein at least part of said at least one outlet port has a hydrophobic surface coating.

In certain embodiments. the resonant beam members of the multi-beam resonator are provided in a parallel arrangement and are fixed at each end of their longitudinal length to a base substrate.

In certain embodiments the at least inlet port allows the test sample to be introduced into the reaction chamber from the top of the reaction chamber.

In certain embodiments at least part of the upper and lower surfaces of the at least one outlet port are coated with a hydrophilic material.

In certain embodiments, the at least one outlet port has a depth of 9Opm and a width of around 06mm.

In certain embodiments, the term monitoring a change" means continuously monitoring the progress of a chemical reaction within a test sample, but taking a plurality of readings which can be used as data to calculate whether a change in density and/or viscosity is occurring within the fluid sample. Typically the resonant beam members are placed in direct contact with the test sample which is undergoing a chemical reaction; typically by immersing the muItibeam resonator in the test sample.

In certain embodiments, the multibeam resonator comprises 3 resonator beam members. As defined herein, a uresonant beam member" is a beam member which is part of a multibeam resonator apparatus which is of specific dimensions and which is further comprised of a material which is suitable for resonating when immersed in a fluid sample.

In certain embodiments, the test strip comprises 2 or more vibratory beam members, typically 3, but also 5 or 7 or more vibratory beam members.

Where 2 or more vibratory beam elements are used, a sheadng effect is used to mediate movement of the vibratory beam member through the fluid sample. Typically said resonant beam members can be provided in a parallel arrangement and can be fixed at each end of their longitudinal length to a base substrate. In certain embodiments, at least one end of the resonating beam members may not be fixed to a base substrate. In further embodiments; the resonating beam members are defined from the same piece of material which forms the base substrate, and hence are therefore integral to the base substrate material.

Without wishing to be bound by theory, the inventors have identified that an arrangement of resonant beam members which comprises 3 resonant beam members joined at both ends confers particular advantages as the moments of the clamped ends of the resonant beam members are cancelled out. Surprisingly; this arrangement has been shown experimentally to have particular advantage when performing measurements in liquids over single ended resonating beam structures which in prior art are in general heavily damped in liquids with density greater than Ig/mI. The benefit observed with a 3 beam resonator should also apply to multi-beam resonators comprising 5, 7, 9 or any other odd number of resonant beam members, which comprise outer resonant beam members positioned about a central beam member.

Typically the resonant beam members of the multi-beam resonator are arranged in parallel configuration, with a central beam and outer beams provided thereabout.

In certain embodiments, the central (or middle) resonating beam is of a first set of dimensions, in particular a first width and first length, while each of the further resonating beams, which are typically arranged in equal number either side of the central beam member in a parallel configuration, are of symmethcal dimensions of longitudinal length and width, along an axis defined by the longitudinal axis in the centre of the central beam.

In certain embodiments, the central resonant beam member of the multi-beam resonator has a width in the lateral dimension of 2mm or less. In one embodiment, the width of the central resonant beam member is about 1mm. In certain embodiments, typically the width of the central beam member is equal to the width of the outer beam members. In certain embodiments, typically the width of the central beam member is greater than the width of the outer beam members. In one embodiment, for a 3 beam resonator, the central beam is the width of the sum of the outer beam widths, said outer beam members typically being positioned parallel to the central beam member, with an equal number at each side of the central beam member.

In certain embodiments, the length of the of the resonant beam members in the longitudinal dimension can be 18mm or less, 16mm or less or 14mm or tess. n one embodiment, the ength of the resonant beam members is 55mm or ess.

n certain embodiments, the distance of the spacing between resonant beam members which are arranged immediatey paraD& to each other is 2mm or tess. n one embodiment, the spacing between the beam members is 025mm or ess.

n certain embodiments the intema height of the reaction chamber is greater than 1mm. n certain further embodiments, the interna height of the reaction chamber is ess than 1 mm, so as to provide a reaction chamber with as smaD a voume as possibe.

n certain embodiments, the distance of the spacing between the outer bngitudina atera side of the outermost resonant beam member and the surrounding housing is 2mm or ess. n particu'ar embodiments where the muftibeam resonator is used to make a measurement of viscosity by way of determining a change in quaDty factor, the dimension of the spacing between the outermost resonant beam and the surrounding housing is 05mm or ess. n particuar embodiments where the mutibeam resonator is being used to obtain a measurement of the density of a sampe fluid, through the use of a change in frequency, the distance of the spacing between the outermost atera surface of the outermost ongitudina beam and the surrounding housing is 0.5mm.

n certain embodiments, the distance of the spacing between the outer atera side of the outermost resonant beam member and the innermost wall of the surrounding housing is greater than 25pm.

In certain embodiments, the total length of the multi-beam resonator of the invention is 20 mm or less. The total length of the multi-beam resonator is typically defined by the summation of the longitudinal length of the resonant beam member and the length of the mounting zone. In certain embodiments, the length of the multi-beam resonator in the longest dimension is around 114mm.

The specificity of the multi-beam resonator to the detection of density and/or viscosity of a sample may be determined by calculating the ratio of the longitudinal length of the resonant beam in view of the length of the mounting zone of the multi-beam resonator. In certain embodiments, the ratio of the beam length to mounting zone length is in the range of I to 9.

In particular embodiments, the ratio of the longitudinal length of the beam to the length of the mounting zone is around 4.

In certain embodiments, the multi-beam resonator is configured with appropriate dimensions of resonator beam length, resonator beam width, and with a specific distance between the parallel beam members and the outer housing such that mode I (the first mode of vibration of the resonant beam member) and/or mode 3 (the third mode of vibration of the resonant beam member) can be used. In particular embodiments, mode 3 (the third mode of vibration) is preferred.

In a particular embodiment, the multi-beam resonator according to the present invention would comprise: 3 resonant beam members, having a longitudinal beam length of 18mm or less, and a central beam width of 2mm or less, wherein the distance of spacing between the 3 parallel resonant beam members is 015mm or less, and wherein the ratio of the longitudinal beam length to the mounting zone is in the range of I to 9. In such an embodiment, the resonant beam members are typically 5.5mm bngitudina beam ength, wherein the ratio of the bngitudina beam ength to the mounting zone is between 1.59 and 4.67.

TypicaDy the resonant beam members are formed from a base substrate materiaL Afternativey, the resonant beam members are conjoined to a base substrate which defines part of the mufti-beam resonator device, and which provides structura support in terms of strength and rigidity.

An area of the base substrate which runs substantiafly paraUe to the resonating beam(s) may be known as a frame members, or housing. Said frame members are typicaHy positioned parafl& to either side of the outer periphery of the mufti-beam resonator. n certain embodiments, the frame members are of dimensions which differ to the dimensions of the resonating beam members. n particu'ar, the frame members can have a different ongitudina ength to that of the resonating beam members, this being necessary in order to prevent stray resonances from interfering with the resonating beams, as such interference may resuft in a reduction in the sensitivity and specificity of the measurement of the properties of the resonating beam members when in use. n certain embodiments, the frame members are of a ongitudina ength which is greater than the ongitudina ength of the resonating beam members, so as to produce an ampitude4requency peak which is at east 200 Hz ower than the amphtude4requency peak of the resonating beams.

n certain embodiments, each resonating beam member is capabe of being resonated at a frequency of between about I kHz to about 500kHz.

n certain embodiments, the muftibeam resonator device may comprise a reaction chamber which receives and retains the test sampe. n such an embodiment, the resonant beam members are positioned within the reaction chamber in such a way that at least one of them is immersed in the test sample when it is provided in the reaction chamber, For the avoidance of doubt, a hydrophobic or hydrophihc coating as described herein may be substituted by a material that displays hydrophobic or hydrophilic properties or a material modified, for example but not limited to a plasma treatment, to display such properties.

In certain embodiments, at least part of the inner surface of the reaction chamber, which for the avoidance of doubt may include any of the surfaces of the resonant beam members, may be coated with a hydrophobic coating to repel a liquid test sample from being present in part of the volume of the reaction chamber, In certain further embodiments, at least part of the inner surface of the reaction chamber, which for the avoidance of doubt may include any of the surfaces of the resonant beam members, may be coated with a hydrophilic coating which promotes the entry of a liquid test sample into the reaction chamber.

In certain further embodiments, parts of the inner surface of the reaction chamber, which for the avoidance of doubt may include any of the surface of the resonant beam members, may be coated with a hydrophilic coating which promotes a liquid test sample to enter in part of the volume of the reaction chamber and yet other parts may be coated with a hydrophobic coating to repel liquid from part of the volume of the reaction chamber.

In certain embodiments, the surface of at least part of the reaction chamber can be provided with a wettable, nonreactive coating.

In certain embodiments, the reaction chamber defines a volume. Typically this volume is static and allows a defined amount of test sample to be retained within the chamber, Retaining a set amount of sample within the reaction chamber allows a set amount of reagent to be added to the sample in order to promote the occurrence of a chemical reaction. In certain embodiments, the internal volume of the reaction chamber is equal to, or less than I000p1.

Typically the reaction chamber is comprised of a plurafity of layers, wherein lower and upper layers define the base and lid of the reaction chamber respectively, and wherein these layers are comprised from or coated with a hydrophflic material.

In certain embodiments the reaction chamber further comprises a stainless steel layer from which the resonating beam members are defined.

In certain embodiments, the reaction chamber includes an opening above the resonating beam members that is substantially open to the reaction chamber volume, permitting for example, but not limited to, application of sample or escape of air from the reaction chamber.

In certain embodiments, a means of substantially covering the upper opening, or top of the reaction chamber volume above the resonating beam members is provided, herein and is in particular described as a reaction chamber lid structure.

In certain embodiments, a means is provided to allow entry of a test sample into the reaction chamber in the form of at least one inlet port.

Typically this inlet port is provided within the reaction chamber lid structure. Typically an in'et port will be mounted above the reaction chamber to aid filling, for exampe by expoiting the effect of gravity.

n certain embodiments the inet port is designed to accommodate the method of appllcation of the test sampe, for exampe by using a pipette whereby the dimension of the inet port permits entry of the hquid sampe, but prevent the pipette tip coming into contact with the inner surface of the reaction chamber.

n certain embodiments, the reaction chamber may be connected with at east one outlet port in order to allow air to vacate the reaction chamber upon bading of the reaction chamber with test sampe. Typically an outlet port has a smaller opening area than an inet port.

n certain embodiments at east one outlet port is provided above the reaction chamber to permit air to escape. n certain embodiments, an outlet port is positioned radially furthest from the at east one inet port. In certain embodiments an outlet port is positioned in a comer of the reaction chamber.

In certain other embodiments, at least one outlet port is located below the resonant assembly to allow air to vacate from below the resonant assembly as the reaction chamber fills with Uquid. The inventors have surprisingly identified that an outlet port positioned below the level of the sample, which is constructed such that it enables air to escape, but retains the liquid sample within the reaction chamber, can be used in the apparatus of the present invention. In a particular embodiment where a reaction chamber is constructed with an intemal volume of about 60p1 with 2 outlet ports positioned below the each end of the central resonating beam members having dimensions 0.6mm by 0.09mm, it was surprisingly observed that no liquid sample was observed to escape from the outlet ports when the device was placed on an adsorbent surface.

In certain embodiments, the at least one outlet port is positioned to pass through at least one side waR of the reaction chamber, optimally at the lowest point above the base layer of the reaction chamber. Typicafly at least part of the inner surface of the outlet port may be coated with hydrophobic material. In certain embodiments, 2 outlet ports are provided, optimally positioned longitudinafly below the centre resonating beam structure.

In certain embodiments, the at least one outlet port is positioned to pass through the side wall of the reaction chamber, optimally at the highest point below the reaction chamber lid. Typically at least part of the inner surface of the outlet port may be coated with hydrophobic material. In a further embodiment, 2 outlet ports are provided, optimally positioned longitudinally above the centre resonating beam structure.

The inventors have further identified that the combination of at least one of: an inlet port, an outlet port and a reaction chamber with a defined volume, permits the precise and accurate loading of a liquid sample into the reaction chamber without the need for external metering dispenser, for example by Gilson" digital pipette. The inventors have identified that this is particularly advantageous for ensuring consistency when using the apparatus of the invention.

In certain embodiments, the resonating beam members are disposed within the reaction chamber in a position where said resonating beams(s), or more specifically the outer most portion of the most outermost resonating beam is located a defined distance from the walls which define the reaction chamber. Typically, the resonating beams are arranged such that the distance between the inner surface of the reaction chamber and the outer periphery of the resonating beams minimises the shear effect in llquid. The distance between the outer periphery of the atera sides of the beams and the inner surface adjacent walls of the reaction chamber cothd be hard to contro in manufacture. The inventor has identified that in certain embodiments of the invention intended to detect sub-centipoise changes in viscosity, there is no significant interaction with the outer periphery of the resonating beams to the inner walls of the reaction chamber surface where the distance is 0.5mm or greater. In mode 3 with beams moving in opposing directions (to maximise shear), a distance between moving surfaces between the beams of 0.25mm yields no significant increase in shear effect. This signifies that the distances required to obtain a significant shear effect between two surfaces could be much lower than 0.25mm. In certain embodiments, the minimum distance between the outer longitudinal periphery of the resonating beams and the inner surface of the reaction chamber wall that is possible to be produced with chemical machining techniques using a 200pm thick part is approximately 200pm. However, finer tolerances are available with thinner materials, such as is achievable using a 100pm thick part, which could have a 100pm outer periphery spacing. A 20 pm thick part could have a 2Opm outer periphery of the resonating beams-inner surface features and so forth. The inventor predicts that the distance between the beams-inner surface where unwanted viscosity effects become prevalent for measurements in liquids with a centipoise value approaching water could be as low as 25pm before the shear (tribologic) effect becomes significant.

In certain embodiments, a reaction chamber is formed around at least one resonant beam member by applying layers of water resistant material to both sides of the resonator substrate assemby, induding but not imited to spacer ayers, water resistant materias, joining or bonding materias, Hd structures, base structures. n certain further embodiments a reaction chamber is formed around at east one resonant beam member from aminates of poymeric materias, such as but not imited to poyester, poystyrene, PEEK, acryc, poycarbonate or from metas such as auminium or stainess steeL In certain further embodiments such laminates may be flat sheets, joined together to form features such as the reaction chamber. In certain further embodiments, the materials may have 3 dimensional features formed into them by techniques such as but not limited to injection moulding, thermoforming, embossing, stamping, punching, partial photochemical etching. A particular advantage of using such a technique may be to reduce production costs by eliminating materials or reduce variabifity of reaction chamber volume between multiple sensors produced.

It is weD known in the art that proteins bind to surfaces that are hydrophobic, such as but not limited to polystyrene, unoxidised metal films. This is a common practise for example in the immobilisation of antibodies onto plastic microtitre plates by merely placing the proteins in contact with the surface. This is a physical adsorbtion process where the proteins partially denature on contact. Often additional proteins need to be added to block" the hydrophobic surface and remove this bias from the results. The inventors have found that the use of hydrophilic materials in the resonator reaction chamber reduces the effect of physical adsorbtion to the reaction chamber walls. The inventors have also found that reaction chambers formed from hydrophilic materials, fill cleanly without trapping air bubble on the surface; enable simple pipetting from a single inlet to completely fill the reaction chamber in a single application without the need for an external metering dispenser (for example a "Gilson" digital pipette). In certain embodiments, materials are plasma treated to promote hydrophilic behaviour. In certain embodiments, hydrophilic materials or coatings are used as part of the reaction chamber construction. In one embodiment, materials forming part or all of the inner surface of the reaction chamber are formed from a hydrophilic material.

In certain embodiments, laminates are fixed together in a way to prevent egress of liquid from the reaction chamber, for example but not limited to adhesive bonding using pressure sensitive double sided tapes, heat activated adhesive, moisture activated bonding agent, liquid gasket.

The inventors have discovered that the height of the reaction chamber has an effect on the quality factor slope or sensitivity. In a particular embodiment where devices with a 8.5mm beam length sensor were made into liquid sensors using spacer layers of various thickness, surprisingly it was found that a sensor with the sum of the thickness of the spacer 203 and 205 more than 0.1mm was not damped. It is well known that the physical amplitude of a resonating assembly will be reduced with the beam length. The inventors predict in a typical embodiment of a 5.5mm beam length, the minimum sum of spacer thicknesses will be less than 10 pm. In a further embodiment of a 14mm beam length, a minimum sum of spacer thicknesses of 0.65mm between the resonator and the base of the reaction chamber is required to resonate and provide a useful response.

Furthermore, the inventors predict that the relationship between resonating assembly beam length and reaction chamber height will not be linear and for a resonating assembly with a beam length of 5,5mm, a useful response will be obtained with a total reaction chamber height of 10 pm or more. Yet shorter resonating assembly beam lengths are possible and for these it is expected that lower reaction chamber height will be possible.

The inventor has identified that in embodiments of the invention which extend to the use of the apparatus or methods of the invention for detecting the occurrence of clotting, coagulation, geflation, agglutination, or precipitation or the like, within a test sample, the reaction can be made to occur in a reduced period of time under conditions wherein the surface area of the multibearn resonator device to volume ratio of the sample is maximised, Achieving an increase in the sensing surface area to volume ratio can be achieved in a number of ways. In one embodiment, the dimensions of the reaction chamber into which the sample is placed can be varied in order to maximise the surface area of the resonator exposed to the sample. In certain further embodiments, the sensing surface area to volume ratio of the sample can be increased simply by reducing the internal volume of the reaction chamber..

In relation to embodiments of the invention which test for the presence of endotoxin within a test sample, which may typically occur upon the interaction of amebocyte lysate and endotoxin, the inventors have observed that the traditional gekclot LAL-based analysis can take up to a time of 1 hour for gellation to occur to the sample mixture. Using the methods of the present invention, the inventors has observed that reducing the internal volume capacity of the reaction chamber, such that the amount of sample contained therein is 6Opl, can significantly reduce the gellation time for samples which are known to have endotoxin present therein.

Furthermore, the inventors predict that further reducing the volume of the sample chamber will serve to further reduce the time required for gellation.

Such a principle would also apply to any other reaction wherein a gellation or similar was to occur within a test sample. For example, without being bound by theory, the inventors predicts that varying the dimensions of the reaction chamber, such that it defines an interna voume of from about 5 p to about 10 p woud aHow geDation to occur within a time period of ess than 60 seconds, this being based on a sampe where endotoxin was present therein. Providing screening methods wherein a quantitative resuft can be provided in r&ation to the presence or otherwise of an anayte or contaminant, such endotoxin, in a sampe within 60 seconds represents a considerab'e, and unexpected advance over similar

techniques known in the prior art.

n certain embodiments, the samp'e is a fluid, in padicuar a Uquid and enters into the reaction chamber through the inet port by means of capiflarit'y.

n certain embodiments, at east one surface of the reaction chamber comprises a reagent which may promote the occurrence of a chemica reaction, such as, for exampe, amebocyte ysate or a synthetic anabgue thereof in order to promote a geflation reaction in a test sampe which contains endotoxin. n certain embodiments, at east part of at east one surface which defines the interna voume of the reaction chamber can be coated with a reagent, for exampe amebocyte ysate or a synthetic an&ogue thereof, in the case of an endotoxin detection assay.

n embodiments of the invention where the methods are used to determine the presence of endotoxin in a test sampe, the amebocyte lysate can be dried onto a surface within the reaction chamber, using techniques such as yophHisation. n certain embodiments, the dried amebocyte ysate can be present as a coating or deposit on at east one inner surface of the reaction chamber of the test strip or on one or more surfaces of the resonator within the reaction chamber. n embodiments, where dried amebocyte lysate is provided within the reaction chamber, typically the amebocyte lysate is reconstituted following the addition of a liquid test sample. For example, in certain embodiments, the ingress of the liquid sample into the reaction chamber results in the dried amebocyte lysate being reconstituted. Once reconstituted, the amebocyte lysate can participate in the reaction with any endotoxin present in the sample.

In certain embodiments, the reagent, such as amebocyte lysate, can be provided in a Uquid solution, and can be added to the reaction chamber, prior to, simultaneously, or following the loading of the reaction chamber with the test sample. In certain embodiments, the test sample can be mixed with the reagent, such as amebocyte lysate, to form a test sample mixture prior to loading the test sample mixture into the reaction chamber.

The reagent, such as amebocyte lysate, may be coated onto a surface of the reaction chamber using any suitable coating technology which is known to the person skilled in the art, which is know for the purpose of coating at least part of a with a reagent, such as amebocyte lysate.

Examples of such coating technology include, but are not limited to; screenprint, dropdeposition, dip coating and inkjet techniques.

The inventors have surprisingly discovered that the amount of reagent dispensed onto a beam can be quantified by measuring at least one parameter associated with the vibration of the resonant beam member, including frequency, amplitude, quality factor and phase in air.

Typically, the resonant beam members are composed of a material which allows them to resonate or oscillate. In certain embodiments, the material from the beam structures are substantially composed is an inert material.

In certain embodiments, the resonating beam members are composed of a material selected from the group consisting of, but not limited to; silicon, alumina, aluminium, copper, palladium, iron, gold, platinum and steel. In embodiments where the beam members are comprised of steel, typicafly this is stainless steel.

In embodiments where the beam members are provided as an integral part of the base substrate, said beam members may be formed by a technique selected from, but not limited to; etching, in particular photochemical etching, laser treatment and mechanical punching of the base substrate. In other embodiments where beam members are not provided as an integral part of the base substrate, said beam members and base substrate may be formed by but not limited to any of the above techniques and joined together using a technique selected from but not limited to: adhesive bonding, welding, mechanical assembly, soldering.

In certain embodiments, the vibratory element which mediates oscillation of one of the beam members is a piezoelectric element. The piezoelectric element, which may also be referred to as a piezoelectric actuator, may be conjoined to at least one resonant beam member at any suitable position which can result in oscillation of the resonant beam member upon excitation of the vibratory element. The piezoelectric material can be electrically connected. Typically the piezoelectric element serves to cause oscillation of at least one of the resonant beam members, typically at a fundamental frequency of that beam member. In certain embodiments, the piezoelectric material can mediate oscillation of at least one of the resonant beam members at a harmonic frequency.

The piezoelectric material may be any suitable piezoelectric material known to the skilled person in the field, and may in particular be selected from the group comprising, but not limited to a polymer such as PVDF (polyvinylidenedifluoride), a crystal or a ceramic material. En certain further embodiments, the piezoelectric material is PZT (lead zirconate titanate).

In certain embodiments, the PZT is provided in the form of a screen printed PZT actuator.

In such embodiments, the apphcation of an electrical signal or electrical power to the piezoelectric material results in the vibration of the piezoelectric material and in turn the vibration of the conjoined resonant beam member, or of at least one beam member which is located near to the piezoelectric material in cases where the piezoelectric actuator is provided upon the base substrate as opposed to the actual resonant beam. This vibration may alternatively be referred to as actuation of the beam member.

In certain further embodiments, the vibrational movement of the beam member can be induced by magnetostriction or by direct magnetic actuation mediated by magnetic shape memory materials, Accordingly, in certain further embodiments, the vibratory element may be magnetic shape memory materials. Such materials include, for example, ferromagnetic shape memory (FSM) alloys which exhibit large changes in shape and size upon application of a magnetic field.

In certain further embodiments, vibration of the beam member may be achieved by a vibratory element which is provided in the form of a transducer which converts electrical energy into kinetic energy in the form of a resonance vibration at a specific frequency. The transducer may be connected to any suitable electrical energy source. A connecting means conjoins the transducer to the beam member allowing the kinetic energy to be transmitted from the transducer to the beam member, this resulting in vibration of the beam member.

In certain embodiments, eectrica contacts and/or connections connect the vibratory dement to an extema contrd unit. These dectricS connectors function to suppy &ectrica power to the vibratory dement. n certain embodiments, an insuating ayer may be provided over the &ectrica contacts to prevent short circuiting.

TypicaUy the vibratory &ement causes at east one of the beam members to resonate in a transverse direction. The phase of the resonation is a function of the vibration mode. The vibration mode can be sdected by exciting the vibratory dement (resonator) within a predefined frequency range. Different resonant modes are achieved by s&ecting a different energy state. Typicafly, the strongest energy state is sdected, said state being out of phase by exciting the resonant beam at a particuar frequency. n certain embodiments, the beam members of the test strip can be excited by the use of pink noise) white noise, a chirp or the Uke.

This can) in certain embodiments) then be used as basis to determine the resonant mode 1, resonant mode 3 or any other usefu mode of the resonant beam member. n akernative embodiments) the Q$actor can measured the response resutting from excitation the chirp, pink noise or white noise response. n certain further embodiments) dosed oop osciflation of the at east one resonant beam member may be used. n certain other embodiments chirp, pink noise or white noise can be used to generate a signa' from which the resonating beams can be cahbrated.

In certain embodiments, oscillation of a resonant beam member can be detected by a sensor means or sensor dement. In a particu Oar embodiment, where a vibratory element is attached to at least one resonant beam member of the muItibeam resonator device) the sensor means is conjoined to a different resonant beam member, In certain embodiments, where a vibratory element is attached to at least one resonant beam member of the multibeam resonator device, the sensor means is conjoined to the opposite end of the same resonant beam member. In embodiments where the vibratory element is conjoined to the base substrate, as opposed to a specific resonant beam member, the sensor element may be conjoined to a resonant beam member, or altematively, may be applied to the base substrate, in the proximity of the resonant beam members, In further embodiments, a plurality of sensor means may be used. Said plurality of sensor means may be applied to different beam members, to the base substrate or to both the resonating beam members and further to the base substrate.

In embodiments where the sensor means is applied to the base substrate, typicafly said sensor is applied to an area which is proximal to the end of the length of the beam member. Typicafly, the sensor means is conjoined to the beam member in proximity to the end of the beam members, but at a longitudinal end opposite to that where the vibratory element is provided.

This spatial separation of the vibratory element and the sensor element (which may also be achieved by conjoining the vibratory element and the sensor element to different beam members) ensures that the sensor detects only oscillation of the beam member, and is not influenced directly by the vibration which is being emitted from the vibratory element.

In certain embodiments, the sensor means is a piezoelectric member, or substantially comprised of a piezoelectric material. This piezoelectric element serves to detect and convert the physical movement of the beam member to which it is conjoined into a measurable signal. The sensor means may further have electrical connectors conjoined thereto, in order that an output signal can be sent to a unit which can use the output signal as a measurable signal in order that the oscillation of at least one of the associated beam members can be determined.

Typically, the sensor means for determining the frequency of oscillation of at east one of the beam members is conjoined directly to a beam member, Generally, said beam member is also dfrectly conjoined to a vibratory element. In certain embodiments wherein there are 3 or more beam members, sensor means for determining the frequency of oscillation may be conjoined to more than one of said beam members, said beam members typically being a beam member which does not have a vibratory element conjoined thereto. Typically however, both the vthratory means and the sensor means are attached to the same beam member, with these being spaced at either ends of the beam member.

In certain embodiments, electrical contacts and/or connections connect the sensor means for determining the frequency of oscillation to an external control unit. Typically the piezoelectric actuator and piezoelectric sensor elements and electrical connections thereto are insulated from the sample fluid which is present in the reaction chamber.

In various further aspects of the invention, there is provided a test strip which comprises the multi-beam resonator device of the invention. Such a test strip can be used for the automated analyss of the density and/or viscosity of a test sample.

As such, a yet further aspect of the invention provides a test strip comprising multi-beam resonator device as herein defined, for use in the quantification of the occurrence and progress of a chemical reaction within a test sample, by means of monitoring a change in the viscosity and/or density of a test sample which is undergoing a chemical reaction, after a defined period of time wherein said chemical reaction may result in the geflation, agg'utination, precipitation or coagutation or the like of the test sample.

As such, a yet further aspect of the invention provides a test strip comprising multibeam resonator device as herein defined, for use in the continuous, reakime monitoring of the occurrence and progress of a chemical reaction within a test sample, by means of monitoring a change in the viscosity and/or density of a test sample which is undergoing a chemical reaction, wherein said chemical reaction may result in the geHation, agglutination, precipitation or coagulation or the like of the test sample.

In various further aspects, the present invention extends to the use of the foregoing methods and apparatus in methods for monitoring the progress of assay methods and chemical reactions, by way of monitoring a change, or otherwise, of the density and/or viscosity of a test sample.

Accordingly, a further aspect of the invention provides for the use of a method as hereinbefore described for the continuous monitoring of a test sample in order to determine a change in the viscosity and/or density of the test sample, wherein the sample is, or may be, undergoing a chemical reaction.

In certain embodiments, the method may be used for the detection and/or quantification of a contaminant, such as bacterial endotoxin, in a test sample, said test sample typically being a fluid sample.

In certain further embodiments, there is provided the use of a method according to the invention for monitoring a change in the density and/or viscosity of a test sample, wherein said test sample may be used in an immunoassay, such as an ELJSA, or in an aggkitination assay, or in an assay to determine coaguaUon of the test sampe.

A yet further aspect of the invention provides for the use of a mufti..beam sensor apparatus, as hereinbefore describe, for use in determining the density and/or viscosity of a test sarnpe, wherein the sampe is, or may be, undergoing a chemic& reaction.

n certain embodiments, the test sampe is a test sampe which is to under go a coaguation reaction, an immunoassay, such as an ELSA, or an aggutination reaction.

n certain further aspects, the invention extends to the use of the test strip of the present invention in the continuous monitoring of a change in the viscosity and/or density of a test sampe which is undergoing a chemica reaction wherein the chemica reaction resufts in the formation of a geilation, agg'utination, coaguation or precipitation product or the Uke. n certain embodiments, such a chemica reaction r&ates to the formation of a ge during the performance of an endotoxin detection assay, such as a Limu!us amoebocyte ysates (LAL)based endotoxin screening assay. n certain further embodiments, the chemica reaction is that which occurs during the performance of an immunoassay, such as an ELSA assay. n further embodiments, the chemica' reaction is that which occurs during a coaguation assay, such as the determination of coaguation time as part of a prothrombin test. n certain further embodiments, the chemic& reaction r&ates to the formation of agg'utination complexes during a latex agglutination assay, or similar According to a yet further aspect of the invention there is provided a kit for use in monitoring a change in the viscosity and/or density of a test sample through the determination of at least one data value which is derived from a resonant beam member which is caused to resonate within the test sample, said data parameter being used to calculate the density and/or viscosity of the test sample in order to determine whether the test sample is undergoing a chemical reaction, the kit comprising a test strip according to the invention abng with instructions for the use of the same and the provision of appropriate reagents.

In certain embodiments, the occurrence of the chemical reaction within the sample mixture results in the formation of a geDation, aggOutination, coagulation or precipitation product or the like within the sample mixture.

According to a yet further aspect of the invention, there is provided a test kit for detecting bacterial endotoxin, said kit comprising: -a test strip comprising at least 3 resonant beam members which are provided within a reaction chamber having a defined internal volume and which is suitable to receive and retain a test sample, a reagent comprising amebocyte lysate or a synthetic analogue thereof, -means for causing at least one of the resonant beam members to resonate and means to detect the resonation of at least one of the resonant beam members, and -instructions for the use of the same.

In certain embodiments, said kit further comprises apparatus and/or instruments for performance of the method which are sterilised such that they are free from endotoxin contamination.

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the

art in the field of the present invention.

Throughout the specification, unless the context demands otherwise, the terms compris& or include', or variations such as comprises' or comprising', includes' or including' will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

As used herein, terms such as "a", "an" and "the" include singular and plural referents unless the context cleaiiy demands otherwise. Thus, for example, reference to "a resonant beam member" includes a single resonant beam member as well a two or more different resonant beam members,

Brief description of the drawings

Figure 1 shows a representation of a test ship and a test meter and the stepwise process used to calibrate the test meter and confirm the viability of the test strip (Figure 1(a)), the loading of the sample to be tested onto the test strip (Figure 1(b)), and the engagement of the loaded test strip with the test meter in order to allow analysis of the test sample, and the provision of an associated reading (Figure 1(c)), Figure 2 shows a schematic representation of a test strip (Figure 2(a)) as well as an exploded version of said test strip (Figure 2(b)), Figure 3 shows an alternative embodiment of a resonant assembly which may be used in the method of the invention (Figure 3(a)), along with an exploded view thereof (Figure 3(b)), Figure 4 shows a typical triple beam resonator sensor response to a gellation or coagulation reaction: this shows a LAL and Endotoxin (IOEU/ml) reaction, Figure 5 shows a typical triple beam resonator sensor frequency response to temperature, Figure 6 shows the parameters of the triple beam resonator which were varied in order to asses the correlation of sensor dimensions to viscosity and density measurement sensitivity, Figures 7 (a) to (f) show a series of tables illustrating the dimensions of 6 different families of triple beam resonators, Figure 8 (a) shows the FEA for a triple beam resonator design (cell 2 DOE) at mode I at 16,436 Hz, while Figure 24(b) shows the triple beam resonator (cell 2 DOE) at mode 3 at 18347 Hz, Figure 9 shows a graph illustrating density/viscosity sensitivity of triple beam sensors comparing resonant modes against frequency response at resonant modes I and 3, Figure 10 shows a graph illustrating density! viscosity sensitivity of triple beam sensors comparing resonant modes against 0 (quality factor) response at resonant modes I and 3, Figure 11 shows a graph illustrating the relationship between the ratio of the beam length to beam mounting zones versus performance of triple beam sensors using frequency response slope with viscosity! density standard fluids, Figure 12 shows a graph illustrating the r&ationship between the ratio of longitudinal beam length to the ength of the beam mounting zone versus performance of triple beam sensors using quality factor response sbpe with viscosity! density standard fluids.

Figure 13 shows a graph illustrating the relationship between the outer beam to frame distance versus performance of triple beam sensors using quahty factor response slope with viscosity! density standard fluids.

Figure 14 shows a graph illustrating the relationship between sensitivity to viscosity standards against distance between the moving beams, Figure 15 is a table showing the dimensions of the triple beam resonators as well as their results in terms of viscosity and density sensitivity, the designs Usted correlate with the sensors detailed in Figure 7, for example Cell 15 DOE is the same as design 15 as detailed in Figure 15, Figure 16 shows a bar chart relating sensitivity to viscosity standards for high performing triple resonator designs as shown in Figure 7 (for example Design 33 is equivalent to Cell 33 DOE of Figure 7 and further to the designs listed in Figure 15), where the devices are classified on the basis of the viscosity sensitivity based upon the &ope of the Q-factor versus the standard concentration curve, Figure 17 shows a bar chart iflustrating sensitivfty to density standards for high performing trip'e resonator designs identified according to Figure 7 (for exampe Design 21 is equivaent to CeO 21 DOE of Figure 7)and further to the designs isted in Figure 15, where the devices are classified on the basis of the sensitivity based upon the sope of the frequency versus the standard concentration curve, Figure 18 shows 3 graphs iflustrating the resonance spectra of a singe thpe beam resonator measured Qeft hand graph) before coating with protein, (midcfle graph) after coating in protein, (right hand graph) after treatment with a Trypsin, Lypase and Amyase soution, Figure 19 shows a graph iflustrating the rea-Ume measurement of a protein fflm coating the resonator being removed using with a Trypsin, Lypase and Amyase soution, Figure 20 shows a graph iflustrating a change in Q factor of the anti.phase at 2&5kHz against time, as the protein was removed from the tripebeam resonator by the action of a protein remov& agent, Figure 21 shows the frequency response of the trip'e beam resonating sensor to water (Figure 21(a)), and to air (Figure 21(b)), Figure 22 shows the frequency response of the tripte beam resonating sensor to sugar soutions of 0% and 85% w/v, Figure 23(a) shows a response peak of aOl EU/m endotoxin sampe. Response curves were taken at 80 second interv&s.

Figure 23(b) shows the response peaks of a 0EU/m endotoxin sampe. Response curves were taken at 80 second interv&s, Figure 24 shows that the sensor gives an output that corresponds to the concentration of endotoxin in the water sampe. Q factor changes as the LAL reacts with the endotoxin. (Lines were Eabefled as foDows: smafi diamonds, 0EU/m, boxes/crosses 0.1 EU/ML, spheres 1EU/m, and trianges IOEU/ml), Figure 25 shows that graph iflustrating that a sensor gives an output that corresponds to the concentration of endotoxin in the water sampe. 0 factor changes as the LAL reacts with the endotoxin, and Figure 26 iflustrates the relationship between reaction chamber height and 0 Factor response to viscosity/density standards.

The present invention wifi now be described with reference to the foflowing examples which are provided for the purpose of illustration and are not intended to be construed as being limiting on the present invention.

Detailed Description of the ifivention

The present invention provides an improved apparatus method for the real time measurement of assays and chemical reactions wherein a change in viscosity and/or density, by for exampe, geation, precipitation, aggutination or coaguation, occurs.

Without wishing to be bound by theory, the inventors have surprisingy identified that muftiresonator beam devices, such as trip'e beam resonator devices, exhibit high eves of sensitivity to both viscosity and density of a fluid test samp'e. This sensitivity aVows for high accurate readings to be obtained, which can be obtained in a very short, and ongoing, period of time. This therefore aVows the ongoing monitoring of a chemica reaction within a test sampe, in order to track its progress.

FoVowing extensive experimentation, the inventors have identified that the preferentia mode of the doubeended tripebeamed resonating viscometers provided by the invention in Hquid is mode 3. The use of a tripe beam embodiment of the muftibeam resonator device in mode 3 provides particu'ar benefits such as (i) that the centre beam moves in anti phase to the outer beams, providing maximum shear of the fluid being samped, conferring superior viscosity sensitivity compared to other resonators, (ii) that the moments at the beam ends are canceHed, ensuring superior signa gain which potentiaHy provides arger dynamic range than other types of resonating sensor for fluid viscosity sensing and (iii) the net mass movement at aD time is zero, resuEting in a sensor that requires ow power to drive, such as that afforded by printed fim PZT or PVDF fflm, yet produces a arge gain that can be easDy read by simp'e and inexpensive e'ectronics.

Furthermore, with regard to the 6 edges of the respective 3 beams gVding through the liquid, the observed high eve of energy efficiency provides high sensitivity to viscosity changes in the surrounding test sampe. Such sensitivity has been identified by the inventors as being particuarIy suitable for monitoring the small changes seen in viscosity and density which occur during chemical, reactions. This is coupled with the ability to monitor the progress of the biological reaction in a rapid and on-going manner in order to provide a novel, field leading approach to assay monitoring.

Furthermore, with regard to the double ended triple beam arrangement, the inventors found that the highly efficient resonating structure particularly suitable for providing a readily measurable output in a wide range of viscous liquids, ranging from that of water to a blood dot. Such a wide dynamic range has been identified by the inventors as being particularly useful for monitoring reactions where the initial state of the reaction mixture is water-like and the end-point is a gel.

Without wishing to be bound by theory, the inventors have identified that the double-ended triple-beamed embodiment of the multi-beam resonator (which may also be referred to as the multi-beam viscometer, or multi-beam microviscometer) provides a highly sensitive viscosity measure with a signal that is strong enough to be readily and easily quantified without complex models using low-cost and portable equipment. For example, and again without wishing to be bound by theory, the inventors have identified that the triple beam resonator devices of the invention provide a response which can be on average up to 60 decibels above the signal noise, this offering hundredths of a centipoise sensitivity.

Furthermore, the triple beam resonator devices of the invention require only a single pair of actuator/receiver elements, situated on one of the beams to obtain a single peak due to an anti-phase resonance. This differs significantly to single ended tuning forks that need moment cancelling actuation or, or double-ended twin-beamed devices that require phase controls. This arrangement as appfled to the triple beam resonator of the invention removes the need for such complex drive electronics and allows for significant simpflfication and miniaturisation of circuitry and interconnections.

Without wishing to be bound by theory, the inventors have further identified, that in certain embodiments, the double-ended triple-beam resonator of the invention produce an improved technical effect when measuring assays as the resonator probes at least SOpm of the liquid test sample located on either side of the resonating beam members. No labels, probes or "beadC are required to facifitate the obtaining of a test result. The arrangement of the beams within the double-ended triple-beam resonator of the invention places the resonating portion at the centre of the reaction, this further helping to achieve maximum sensing effect.

Another key benefit of the resonator device assembly of the invention is the temperature-frequency bias. Vibration based viscometers known in the art, such as that disdosed in US 521 15054, provide an output signal which is greatly dependent upon temperature. The double-ended triple-beam resonator device of the invention is affected by only minimal temperature to signal output deviations. Such derivations have in fact been identified as being predictable, hence they can be taken into account when analysis any results produced.

The multi-beam resonator devices of the invention can be easily waterproofed and therefore can be electricaily insulated, such that they have improved functionality when used with fluids having high dielectric constants. Resonant sensors known in the prior art are typically damped at the mountings, such that a waterproof coating is not typically used. If a coating is used, this must be kept thin, possibly as this as lOOnm, however capacitance coupling between the electrodes and the liquid occurs and drowns the resonant response. This does not occur with the multi-beam resonators of the present invention, and as such layers as thick as bOurn can be apphed without damping the resonance.

Due to these properties, the apparatus and methods of the invention may have particular appUcation in such diverse areas as haematology, biochemistry, immunology, microbiology, DNA analysis, protein crystalisation, ceO cultures and more generally in any high throughput screening technique, or assay method, such as an immunoassay, which is used for the detection of a contaminant, analyte, antigen or antibody in a test sample.

In one application, the apparatus and methods of the invention may have particular utility in assays for detecting endotoxin contamination of samples, and in particular fluid samples. The present invention uses a multi-beam resonating sensor to provide a quantitative analysis of a LAL assay. The detection of at least one of a number of parameters associated with the oscillation of a resonating beam member which is provided by the multi-beam resonating sensor allows for the dynamic monitoring to determine the occurrence and progression of a gellation-like clotting reaction which occurs following the exposure of horseshoe crab amebocyte lysate to endotoxin, The double-ended triple-beam viscometer of the invention not only provides a highly sensitive, real-time measurement of the on-going LAL based gellation reaction, but also provides a significant advance over LAL-based analytical methods known in the prior art, as the present methods and apparatus do not require a waiting time to allow the gelation reaction to occur before the results can be obtained, Rather, apparatus and methods of the present invention can monitor the progress of the reaction immethatey due to the high sensitivity, hence it is not necessary to waft for the geHation reaction to advanced to a stage where there is entrapment of partides in order to obtain an assay result. This important advance aVows the provision of an endotoxin test method and apparatus with a 2 minute response time. This shou'd be compared to the 1 hour test provided by assay methods of the prior art, such as that described in US 521 I 054 as discussed hereinbefore.

n one embodiment of the invention, there is provided a sensitive, yet robust testing kit which can be used in app Vcations where the LAL gedot based method woud normaVy be used, for exampe in the testing of diaysate so'utions, or for parenta' testing.

A test strip component, which may be disposabe, contains the muftibeam resonating sensor and any required reagents. The test strip component woud be used in conjunction with a metering device or a reader apparatus which wou'd interact with the test strip such that at east one parameter of the resonating beam members, such as frequency, quaUty factor or resonance phase angie can be determined.

An arrangement of such a test strip and reader apparatus is shown in Figure 1. Speciticafly, Figure 1(a) shows a disposabe test strip 100, which has been removed from its protective endosure and which is ready for use. The test strip is orientated into a position which makes it ready for insertion in the test meter 101.

Once inserted the resonator woud be excited and the resonances measured using a standard frequency anayser as anyone skiDed in the art woud know. For exampe, the document of Green (Pubidy disdosed undergraduate Thesis. Brunel University. 1995) teaches that the frequency of resonance in vacuum, air or Uquid can be found and monitoring using either an open-loop or dosed-loop method. An open-loop method is where frequency is scanned between a pre-defined range, for example using a Solatron 1220 Frequency Analyser. A closed-loop method is where the frequency is scanned from DC until a resonant signal is detected. The circuit then locks onto the signal and tracks any changes by using the output signal from the pickup. As the measurement changes, the beam with the pickup resonates at a sflghtly different frequency from the forced frequency. Part of this signal is corrected for the 90 degree phase shift between the beams and fed back to the first beam forcing the vibration frequency, thus the circuit is self-tracking, automatically detecting changes in resonant frequency. For example, an Apollo Universal Counter Timer could be used for this approach along with a suitable power supply.

Upon inserting the sensor into the reader apparatus, a frequency spectrum depicting the frequency of resonance of the resonating beams present in the multiple beam resonator sensor would be obtained, with this frequency determining the resonating of the beams in air. More specifically, this measurement is achieved by exciting the piezoelectric actuator with a range of frequencies and recording the resulting amplitudes of the resonance of the resonating beam members of the device.

The frequency of the different resonant modes and the shape of the "peaks" would be used to calibrate the test strip and would also be used to validate or self-check the multi-beam resonating sensor before use. This calibration step may be particularly required if the test strip has previously been used to test a previous sample, as this calibration step will determine whether any sample has been retained in the reaction chamber of the test strip from the previous use.

if the sensor passes the initial calibration and valldation test protocol, the display 102 on the test meter 101 will prompt the user to add the sample liquid to the reaction chamber of the test strip. Figure 1(b) shows the appflcation of a Uquid sample to the sample entry window 103 on the test strip. Typically the liquid sample uses capitlarity to enter into the reaction chamber of the test strip. Figure 1(c) depicts the reader apparatus meter 101 showing the result of the assay, this result being directiy hnked to whether a change in viscosity due to the gellation reaction has resulted in the sample.

On completion of the assay, the change in viscosity determined by analysis of the resonant frequency spectrum and the amplitude of nodes less favoured by the resonant beam assembly being surrounded by a gel clot is compared with a known set of data stored within the meter (the calibration curve).

Figure 2 shows a further embodiment of a test strip for use in the present invention. Figure 2(a) shows a schematic of a disposable test strip 200.

Figure 2(b) shows an exploded schematic of the disposable test strip 200 shown in Figure 2(a). The disposable test strip 200 comprises a base substrate layer 201 onto which a reagent layer 202 is disposed.

Alternatively, the reagent layer 202 may be provided as a coating which is disposed upon a part or the whole of any internal surface of the reaction chamber. A first reaction chamber forming layer 203 is provided upon the base substrate 201. This reaction chamber forming layer may be formed using a pressure sensitive double sided adhesive tape, punched or cut or by application of screen printing or ink jet printing. Additionally outlet ports 210 may be formed as part of the layer. A second reaction chamber forming layer 205 may be additionaHy laminated over the first reaction chamber forming layer to provide further reaction chamber height, and may be formed using a patterned precast film, or by apphcalion of a screening printing or ink jet printing technique or through the use of a suitable nonreactive polymeric material.

A resonant assembly 204 comprising a plurality of resonating beams, in this case 3, may be laminated over the first reaction chamber forming layer. Additionally, a mechanical spacer, 206 may be included to ensure even lamination of subsequent layers. A further reaction chamber forming layer 207 is disposed upon the resonant assembly 204. A final reaction chamber forming layer 208 is disposed upon the previous reaction chamber forming layer to permit the inclusion of further outlet ports 211 in the upper part of the reaction chamber. Lastly, a polymeric film 209 is laminated onto the top surface of the upper reaction chamber forming layer 208. The polymeric film 209 provides an upper lid on the reaction vessel which protects the underlying structures of the test strip from mechanical damage. The polymeric film 209 also serves to improve the stiffness of the test strip, particularly where the test strip is in an embodiment where it is disposable. Additionally disposed upon the polymeric film, may be features including an inlet port 213 and an outlet port 212.

Alternatively, the first and second reaction chamber forming layers 203 and 205 may be combined where suitable dimensional and material selection permits, as may the third and fourth reaction chamber forming layers 207 and 208. Furthermore mechanical spacer 206 may be incorporated as part of resonator 204 if required. Altemative materials may permit further combinations of elements, for example embossing of polymeric material to form a combined base substrate 201 with first and second spacer layers 203 and 205.

Figure 3 shows an embodiment of a multibeam resonator structure which is suitable for measuring properties such as the density and viscosity of a test sample, in particular a body fluid, before and during a chemical reaction. As shown in Figure 3(a), there is provided a triple beam resonator sensor assembly 300 for integration into a disposable test strip sensor device of the invention.

Figure 3(b) shows an exploded schematic of a triple beam resonant sensor assembly for integration into a disposable test strip. A base substrate 301 is patterned with three resonant beam structures 302, 303, and 304. These beam structures may be formed by any conventional method such as laser or chemical etching or by stamping of the base substrate. A patterned insulating dielectric layer 305 is disposed onto the base substrate 301. The patterned insulating dielectric layer may be disposed thereon by any conventional method such as screen printing or ink jet printing. Altematively, a pre-cast film may be laminated over the base substrate 301.

Pattemed conductive tracks 306 are disposed on the patterned insulating dielectric layer 305. These conductive tracks may be disposed by any conventional method such as screen printing or ink jet printing and can be composed of any suitably conductive and chemically inert material.

A pair of piezoelectric elements 307 are disposed onto the pattemed conductive tracks 306, at a location which is in dose proximity to the central resonant beam 303. A second patterned insulating dielectric layer 308 is disposed to cover the majority of the patterned conductive tracks 306. The second dielectric layer 308 is shorter in length at the end distal to the beams in order to expose the ends of the conductive tracks 306.

The second patterned insulating dielectric layer 308 may be disposed by any conventional method such as, but not Umited to screen printing or ink jet printing. The second patterned insulating dielectric layer 308 functions to cover the conductive tracks 306 in order to allow the printing of a further conductive track (shown in this embodiment as feature 309) upon this layer. Accordingly, a second set of patterned conductive tracks 309 are disposed over the second patterned insulating dielectric layer 308 and the piezoelectric elements, The patterned conductive tracks 306 and 309 may run the length of the base substrate 301, as shown in Figure 3(a), such that an electrical connection can be made between the piezoelectric elements 307 and an external device, such as a meter, by means of any suitable connector (not shown).

A thick polymeric waterproof layer 310 covers the tracks and electrodes and prevents short circuiting and liquid impedance interference when the device is immersed in water.

Figure 4 is a graph illustrating a typical resonant sensor frequency response to a gellation or coagulation reaction. Trace 401 provides the amplitude vs. frequency response with the resonant sensor dipped in un reacted reaction mixture. The resonant mode with a peak at approximately 25.1kHz changes due to viscosity. In a low viscosity fluid, in this case unreacted reaction mixture, the sensor resonates over a small range of frequencies, the quality factor or Q factor (the spread of frequencies associated with a peak at 4 peak height) is high. Traces 402 to 408 provide the amplitude vs. frequency response with the resonant sensor dipped in a reaction mixture undergoing coagulating or a gellation reaction, at one and a half minute intervals, As the solution undergoes reaction (that eventually forms a thin and watery gel in one hour at 37°C), the peak disappears and the 0 factor decreases. The changing solution between the shearing beams changes the nature of the resonance. This appears as a significant decrease in amplitude shown in the series 402 to 408. This response allows for the determination of the viscosity of the gelLation mixture at the start, during and at the end of the reaction. It is also possible to determine the rate at which the fluid mixture gels. Both of these are used to indicate the level of endotoxin contamination in the sample.

Figure 5 shows a typical resonant sensor response with respect to temperature. This relationship is used both to check that the sensor has been equilibrated/calibrated to the correct temperature and to ensure that it has been manufactured to specification, by comparing the position of a particular resonant peak with its expected position at the temperature of the test strip as measured by the test meter. On insertion of the test strip into the test meter apparatus, the test meter is switched on either by the presence of the test strip breaking a light beam, by depressing a micro-switch, or by engaging with an electrical contact which connect the piezoelectric components to the edge of the test strip. Within the meter 101, and close to the test strip connector port, is a temperature measuring device, either a thermocouple or a thermistor. This measures the temperature in the vicinity of the test strip. This temperature reading is used with the frequency response in air to decide whether the sensor is equilibrated and at the correct temperature.

The multiple beam (multi-beam) resonating sensor of the invention confers a number of advantages over the amebocyte lysate based methods which are used in the prior art for the detection of endotoxin, induding; (i) thermal stabifity, wherein the frequency response to a sample temperature is a smafi contribution to the signaL (ii) sensitivity, where the resonant beam is more sensitive to geflation that a quartz crystal microbalance (QCM) device, such that lower reagent concentrations are needed to obtain a result, Viscosity sensitivity is inversely proportional to fundamental frequency. As such, QCM devices operate in MHz domain, whilst the resonant devices described here operate in the kHz frequency range. (iii) Manufacturing, as the resonant beam sensor is made from low cost materials, for example steel rather than poUshed solid crystals. (iv) Liquid volumes, because, as piezocrystals become smafl they become difficult to handle and the signal bias effects due to damping forces increase. Double-ended resonators resonating do not suffer from this issue as the damping forces are cancefled at each end. QCM crystals need to be placed in housing such that the Uquid meniscus has to be an angle to the mounted crystal. This eHminates reflections from the surface of the hquid bouncing back to the crystal surface. (iv) Electrodes. The electronic components in the QCM define the sensing area. The electrodes are in contact with the sample, so coatings are needed. In a resonant beam the electronics are mounted away from the liquid retaining reaction chamber. Triple beam resonators do not need to be mounted at any spedfic angle.

EXAM PLES

Example 1: A method used for a sensor manufacture Sensor manufacture The multi-beam resonating sensor, which in this embodiment is a triple beam resonator comprised of 3 resonating beam members arranged in a paraflel configuration, is comprised of the following materials: (i) Steel of 200pm thickness supplied from Precision Micro Ltd (Birmingham, UK), (ii) Insulation 4924 (ESL, King of Prussia), (Hi) Gold Cermet Ink 8836 (ESL, King of Prussia), (iv) PZT paste (Highland BioSciences Limited, Inverness, UK), (v) Silver Palladium Ink 9912-K (ESL, King of Prussia), (vi) Insulating Dielectric 240-SB (ESL, King of Prussia), (vii) Polystyrene Film, AR9020 (Adhesives Research, Ireland), (viii) Acrylic Adhesive Tape (Adhesives Research, Ireland), (ix) Hydrophilic Film 9971 (3M, Minneapolis, USA), and (x) Medical Grade Polyester (Autotype, Oxon, UK) The multi-beam resonating sensor fabrication procedure was as detailed as follows: Triple beam resonating sensors were patterned into the sheet steel using a standard photo-etching process. The additional components required to drive the resonator were deposited onto the sheet steel base substrate using a thick film process. Insulation 4924 was deposited in such a way as to prevent the conductive tracks of the top and bottom electrodes from short circuiting. The base electrode for the PZT components was printed on top of the insulation using a gold compound 8836. A PZT paste was printed at the ends of the beams. A further gold electrode was printed over the top of the PZT to provide an electrical connection. A silver-palladium track was created using compound 9912-K to connect the gold electrodes to the edge of the sensor enabling push-fit insertion into a "meter" instrument. The pastes were allowed to level, dried and fired as described in the manufacturer's specification. A rubberised waterproofing layer 240SB preventing liquid coming into contact with the electrodes, preventing liquid impedance from becoming a confounding signal.

The devices were polled by applying in excess of IOOV DC current to the electrodes attached to the PZT, whilst the devices were heated to the curie temperature at above 200oC, The insulated sensors were fitted with a flow ceO constructed from laminated tapes. A layer of double sided tape was patterned to leave the beams unhindered, and was used to form a spacer layer between the base of the sensor and a sheet of polyester. A similar patterned piece double sided tape such as was placed on the top surface of the sensor. A final piece of patterned polyester film was used to create a well. To prevent evaporation of the test sample, a piece of 9971 hydrophilic coated polyester was sealed over the reaction chamber.

ExamDle2-Optimisation of triple beam resonator structure and dimensions This example was performed in order to determine the optimal configuration of the triple beam resonator in relation to its sensitivity in use for detecting viscosity and density in a liquid rest sample. This analysis also allowed the identification of the most influential aspects of triple beam resonator design, which enable the real time monitoring of assays and chemical reactions where precipitation, agglutination, gellation or coagulation is a measurable parameter which indicates the occurrence or progression of a chemical reaction.

Ii) Triple Beam Resonator Design Figure 6 shows the parameters of the triple beam resonator which were varied in the performance of this experiment. In this figure, (a) relates to the width of the middle (central) beam of the resonating beam members, (b) relates to the beam length, (c) relates to the spacing distance between beams, (d) is the beam mounting zone, and (e) is a gap between the outer beam and the device housing. The outer beam widths (not marked) are of equal width and equal to 50% of the width of the central beam as described in (a). Figures 7 (a) to (f) show tables illustrating the specific reaction chamber dimensions of 6 groups of triple beam resonators comprising 48 individu& triple beam resonators (named CeO I DOE through to CeO 48 DOE) which were produced for testing in this example.

The dimensions shows are all in millimetres, Families of designs were made and grouped according to the ratio of beam length to beam mounting zone length. Within the family groups, other parameters including gap to frame and gap between beams were varied to aVow investigation of potential interaction of parameters.

When constructing the various triple beam resonators, the standard dimensions shown in Table I were typicafly used. The low, middle and high setting values which are shown relate to a lowest and highest setting, in addition to centre points (or middle setting) for each parameter. These values are then used as part of the experimental design.

Table I -standard dimensions (in millimetres) of features of the beam members of the test triple resonators Low Setting Middle Setting Beam length 5.5 mm 7 mm 8.5mm Beam width 1 mm 2mm (centre) Overall length 8.5mm 10.45mm 12.4 mm Gap between 0.25 mm 0.5 mm 0.75 mm beams Gaptoframe 0,5mm 1mm 2mm When selecting the dimension of the triple beam resonators used in this example, consideration was taken of the practical constraints of the experiments, such as process used and resulting reaction chamber volume. Reaction chamber volume was targeted as a maximum of 200u1.

As it was necessarg to eliminate printing variability, only one set of PZT print artwork was used, this constraint therefore essentially fixing the size and gap between the 2 PZI pads. In turn, the beam length could not be any shorter than the gap between the PZT pads. Furthermore, the beam length could not be any longer than the overafi distance, end to end, for the PZT pads. As such, some elements of the PZT pads reside on the resonant beam members. Groups of dimensions were investigated by FEA, selecting designs that had dear separation of peaks between mode I and 3. If they are too close together, immersion in liquid tends to make them merge into a single broad peak, making analysis difficult. An example of the FEA for triple beam resonator design 2 (cell 2 DPE) is shown in Figure 8(a) and (b), wherein Figure 8(a) shows Mode I at 16,436 Hz, while Figure 8(b) shows Mode 3 at 18,347 Hz. It was noted that some of the elements in the beam mounting zone deflect upon modal analysis, indicating a dimension of 0mm for parameter (d) (relating to the beam mounting zone) would not be feasible. This was confirmed experimentally, by comparing the response of a resonator (cell 39 DOE with beam length 8.5mm) first with a 1.95mm mounting zone, constructed by affixing a frame of 12.4mm around the beams and then with a 0mm mounting zone, by affixing a frame of 8.5mm around the beams. The response with the 12.4mm frame was found to have clear resonant peaks around 11kHz and 26kHz, while no resonant response was observed in the embodiment with the 8.5mm frame.

{ii) Design evaluation Each sensor was fabricated and tested with a range of viscosity and density standards between 0.98-138 cP (centipoises) for viscosity and across a range of I -1.4 for density.

Performance was measured using three methods (i) amplitude of measured peak above baseline (electrical earth), measured in decibels, (ii) frequency slope: the variation in frequency of response in liquid, and (iii) quality factor slope: the variation in quality factor of the response peak in liquid, measured at 3dB below peak frequency.

The sensors were scanned from a low to a high frequency to identify resonant peaks and to determine the mode of frequency. The dominant mode was selected, and the sensor was chaflenged with a range of viscosity / density standard solutions.

QiD Mode analysis From the experimentation performed, identified clear differences in performance according to the dominant mode of the sensor. It is well know in the art that the density of liquid has a damping effect on resonating structures, reducing resonant frequency response. It is also well known in the art that viscosity can reduce the quality factor, by viscous coupling of the beams Comparing the frequency against the concentration response slope shows a clear difference in behaviour between mode I and mode 3. It can also be seen that overall, mode I offers the ability to have higher sensitivity to monitor changes in density than mode 3, although both modes can be used to gain a response. This is illustrated in Figure 9 wherein the performance (in terms of the slope of frequency versus the standard concentration curve) is shown for resonant modes I and 3.

Mode 3 resonance is unique to a triple beam resonator, where the central beam is out of phase with the outer beams providing maximum probing of the sample.

By comparing the quafity factor against the concentration response, a dear differentiation can be observed between mode I and mode 3, this being shown in Figure 10.

Figure 10 shows that mode 3 offers the ability to have higher sensitivity to monitor changes in viscosity than mode 1. Doube ended tripe beam resonators offer the unique feature of a mode 3 resonance whereby the net forces at the ends of the beams caneS out, providing an improved sensor response as the out of phase transverse movement provide maximum shearing of the fluid sampe surrounding the beam members.

By using an apparatus that measures the frequency and quafity factor of a resonant peaks of a tdpe beam resonator, it was possib'e to obtain sensitive measurement of both density and viscosity of the fluid simuftaneouSy.

(iv) Rdationship of beam eqth to beam mountinQ zone en As mentioned previou&y, the design of the trip'e beam resonators used in this exampe were ilmited by the requirement to keep to a singe set of screen printed images in order minimise process impact on the resuJts.

This resuked in the production of 6 families of resonators as previou&y described in Figures 7(a) to (f) and the tabe in Figure 15. These families can be further summarised as per bSow.

Family 1: Beams having a ratio of beam ength to beam mounting zone ength of 1.59 (designs 4, 12, 20, 28, 36, 44). This family of beams showed an acceptab'e density sensitivity, with the frequency Sopes varying between 500 to 1500 over the range. The viscosity sensitivity was however poor, with the quailty factor sbpes varying between 2 to 16 over the range. Mode I responses are the on'y measurab'e peaks. The gap to frame distance does not appear to affect the frequency response. The 1mm width beams perform weV, whfle the 2mm width beams are compet&y dampened by the Hquid samp'e.

Famy 2: Beams having a ratio of beam ength to beam mounting zone ength of 3.67 (designs 1,3,9, 11, 17, 19, 25,27,33,35,41,43). Designs I and 3 provide a high sensitivity to viscosity. 2mm width beams are high'y damped in iquId. 1mm width beams give easy to measure peaks in water. Beams dose to the outer frame give a higher sensitivity viscosity measurement. Devices with beams dose together give high viscosity! density sensitivity. t is worth noting, that this group indudes design I (ce I DOE), which was incorporated into a reaction chamber taking approximatdy 35u sampe voume.

Famiy 3: Beams having a ratio of beam ength to beam mounting zone ength of 4.06 (designs 5, 13, 21, 29, 37, 45). Design 29(2mm centra' beam width design) has exceent sensitivity to density (frequency sope of 3104) and viscosity (quaty factor sope of 264). However, on inspection of the raw data it was found that this was due to 2 peaks merging, meaning it woud be difficuft to dev&op an agorithm based on this performance at a ater date. Design 5 (1mm design), has simfiar performance. A 0.5mm gap is present between the beams for both design 29 and 5. Both sets of resufls have been removed from the anaysis graphs.

Family 4: Beams having a ratio of beam length to beam mounting zone length of 4.36 (designs 6,7, 14, 15, 22, 23, 30, 31, 38, 39, 47). For each design the modes are close together and usually merge in liquid, distorting the measurement of quality factor. An example is device 15 (ceO 15 DOE) where the two peaks merge at increasing concentrations and the quaty factor is artificiafly lowered, increasing the slope of response.

Family 5: Beams having a ratio of beam length to beam mounting zone length of 812 (designs 8, 16, 24, 32, 40, 48). The viscosity performance is marginal: the maximum sensitivity is 82. Density performance is acceptable, producing a maximum frequency slope of 926 over the range.

This geometry creates modes very close together and which are therefore hard to deconvolute without complex algorithms. An extra peak can be observed between the first and third mode which also makes measurement difficult.

Family 6: Beams having a ratio of beam length to beam mounting zone length of 933 (designs 2, 10, 18, 26, 34, 42). The viscosity performance less than 100. The density slope is 400 tol 300 over the range. The beams further from the frame appear to be better for the measurement of density. The beams closer to the frame produce a more sensitive response to viscosity. Very few devices in this group produce a useable response.

These results are summarised in Figures 11, 12, 13 and 14. A table setting out the dimensions of the triple beam resonators, as weD as their viscosity and density value is shown in Figure 15.

(v) Summary

In summary, a range of sensors were manufactured which were constructed according to example 1, but with the dimensions detailed hereinbefore. A number of high performing beam member designs were identified. These specific triple beam resonators are shown in Figure 16 which shows a table of the best performing triple beam resonator devices when assessed with regard to density sensitivity, which is defined in relation to the slope of frequency standard concentration curve. Figure 17 shows the best performing triple beam resonator devices when assessed with regard to sensitivity, which is defined in relation to the slope of frequency versus standard concentration curve.

From this analysis, a list of typical design features for a triple beam resonating structure for use in the present invention were identified for achieving enhanced measurement of density and viscosity: (i) Mode 3 is preferential for high sensitivity of measurement, (ii) Ratio of beam length to beam mounting zone length around 4 appears to be optimal, but a device with a ratio close to one up to a ratio dose to 9 would stifl provide a measurement of some value, (iii) Both mode 1 and mode 3 used together can provide 2 sets of data from one response. A high performing resonating sensor can be made with a low sample volume 35uL Example 3 Biosensor for studying theDerformance of protein removing agents The efficient removal of protein is an issue in many applications induding medical devices and diagnostics, and selecting the optimal agent for removing protein can be difficult without a means of monitoring the progress o' the removal. This is an example where the method of following a biochemical reaction using a triple beam resonator device according to the present invention can be of use.

A model protein (Limulus Amebocyte Lysate) or LAL was used in this example. A triple beam resonator was (design 9 (Cell 9 DOE) from Figure 7 was selected, however other designs could have been selected) and fitted to a reaction chamber as described in Example 1. The response in air was measured, and several characteristics of the peak were measured as wefi as the frequency and the Q4actor.

The trip'e beam resonator was then incubated in a solution of protein LAL in the absence of endotoxin for two hours and allowed to dry in air. The response in air was measured, and several characteristics of the peak were measure to ascertain the amount of damping due to protein coating the beams: the frequency, Q4actor and phase angle.

A solution containing a commerciafly available protein removing agent used for cleaning soft contact lenses (2mg of Trypsin, Lipase, Amylase in 2ml of pyrogen free water) was added to the reaction chamber of the sensor. The reaction was run at 37°C and the frequency response was measured for 12 minutes.

The sensor was rinsed to remove the cleaning solution and dried in air.

The response in air was taken, and several characteristics of the peak were measured, the frequency, Q4actor (quality factor).

Results The third order response of the resonator was used to assess loading of protein onto the beams. This mode causes the resonator beam members to move in a shearing, and as such material deposed between the beam members and on the surface of the beams will impede the motion of the beams.

Figure 18 shows 3 graphs illustrating the resonance spectra of a single triple beam resonator measured (left hand graph) before coating with protein, (middle graph) after coating in protein, (right hand graph) after treatment with a Trypsin, Lipase and Amylase solution.

Initiay prior to protein oading the resonating beam has a 3d order mode at 28.5 kHz with a quaUty factor of 904 and a 25db gain. After incubation with protein for severa hours the device was aflowed to dry overnight.

After this treatment the beams were heaviy damped, and the resonant peak was reduce to a few docibes of gain, and was too ow for the Q factor to be cacuated.

After treatment with a proprietary protein removing so'ution the peak frequency measured in air increased from a few db to over 20db and the Q-factor was cacuated to be 628.

Rea time man torinci and detection of a enzymatic reaction R was possibe to see the action of the enzymes breaking up the protein film that coat on the sensor in rea' time. The concentrated and viscous soution of enzyme preparation pius the protein film caused arge viscous drag on the movement at the point of resonance, this created conditions for an "antiphaso resonance measurement to become possibe.

Figure 19 shows a graph iflustrating the reakime measurement of a protein fim coating the resonator being removed using with a Trypsin, Lypase and Amyase solution.

As shown in Figure 19, the enzymes removed the protein from the surface, reducing the amount of protein coating the beams and enabUng the beams to shear through the Uquid with less resistance as time passed and the reaction occurred. It can be suggested that the amplitude of the antiphase resonance is reated to the amount of energy required by the device at its resonance.

Figure 20 shows a change in Q factor of the antiphase at 2&5 kHz as the protein was removed from the triple-beam resonator by the action of the proprietary removal agent. R was therefore possible to calculate the Q factor of the device with respect to time (as shown in Figure 20), which indicates the rate of reaction between the deposed protein and the enzymes.

The rate at which the Q factor, amplitude or phase angle or any combination of the three could be used to measure the rate of the reaction fora particular agent or reaction condition.

Example 4Determination of Sugar Concentrations The multi-beam resonating sensor, which in this embodiment comprises a triple beam sensor, was made according to the design described in

Example I

Preparation of Sugar Solutions Materials used were household sugar (sucrose (Tate & Lyle)) and distilled water, The solutions were made by dissolving the required amount of sugar in distiDed water with stirring to ensure all the crystals were dissolved.

The frequency response of the sensor was tested at 20°C by measuring the frequency in air and water, as shown in Figure 21(a) (air) and Figure 21(b) (water).

As expected, the peak frequency drops from about 19,449Hz to 17,638Hz (a change of 1811Hz), due to the increased damping on the triple beam afforded by the more dense water.

The 0 factor also decreases from 720 to 137.8 with the change in viscosity and density between air and water 582.

To enable sensor optimisation in future designs it is important that estimates of the viscosity changes are available. The viscositydensity product was cabrated using sugar solutions between 0 and 85% at 20cC, as shown in Figure 22. The related sugar concentrations are shown below

in Table 2.

Sugar conc. Viscosity Frequency! % w/v Density 0/kg) (mPa.S) Hz Q factor 0 996.9 0.993 17,638 137.8 10.625 1032.3 1.33 17,188 74,73 42.5 1155.3 5.045 16,728 64.59 1420.882 135.41 16,489 45.05 Table 2 Sugar concentrations Example 5-Detection of Endotoxn in a test sample The experimentation in this example was performed in order to determine the effectiveness of the concept of detecting the reaction of Limulus amoebocyte lysate (LAL) with endotoxin within a liquid test sample, using a triple beam resonating microviscometer. In particular, the experimentation was used to determine that the sensor can distinguish between 2 different concentrations of endotoxin within a time period of 4 minutes.

(a) Materials and Methods: Instrumentation The following instrumentation was used: Frequency Analyser and Amplifier (Cypher Instruments, London, UK) Whirlimixer (Fisherbrand. UK) QBD2 Heater (Grant, Shepreth, UK) and Therma' bock (A'mond Engineering, Uvingston, UK) Assay reagents: The following assay reagents were used: (i) PyrogentTM Limulus amoebocyte Iysate (LAL) reagent, () Lonza, Wakersvffle, USA. Lot number #GLI 176, (ii) Contro Standard Endotoxin (CSE) raised in E. Coll.

055:B5, (iv) Lonza, Wakersvme, USA. Lot number #GL0983, (v) Lonza LAL Reagent Water (LRW). Lot number #01119129 Lonza, Wakersville, USA.

Lb) Procedure for Ge Cot Testinj The sensors were depyrogenated using techniques well known to those skilled in the art. The CSE reagent was then reconstituted with LRW and dlluted to working concentrations, as described by the manufacturer. A surlactant, Tween 20, was added at 0.05% as this was found to improve the stabillty of the endotoxin standards.

The sensor was "blocked" with LAL and LRW for 20 minutes before being rinsed and stored in the refrigerator prior to use.

The LAL ysate was reconstituted with LRW as per the manufacturers' specification. An aliquot of water containing CSE was mixed with LAL lysate at a ratio of 1:1, and then immediatey dispensed into the sensor well. lmmediatey following the addition of the sampe Lo the well, the anaysis was performed.

The frequency analyser was configured to scan between 20kHz and 30kHz to obtain an initial air calibration reading. The output signa was attenuated by -7.5db to remove any distortion. The test period was set to 23ms per data point, the total run time was 12 minutes. This allows the capture of 1024 data points in 2 minutes. After each scan the analyser was set to mmethately repeat the scan.

(c) Determinption of Endotoxin As shown in Figure 23(a) and 23(b), it is possible to observe the difference in the frequency response and phase curves for the 0.IEU/ml and OEU/ml endotoxin reactions.

In Figure 23(a) it can be seen that during the first four scans, the height and width of the response curve changes somewhat after each 80 second measurement: the peaks are becoming smaller and flatter. The peak frequency is decreasing slightly as the peaks are becoming flatter, Increasing amounts of energy are used to cause the structure to resonate.

In Figure 23(b) the height and width of the response curve change very slowly after each 80 second time point compared to 23(a). The change in the resonant peak size and shape with respect to time reflect the rate of reaction in the reaction chamber.

Figure 24 illustrates the relationship between relative Q factor measured over approximately 400 seconds, and the concentration of endotoxin in the sample. There is a clear definition between the four different endotoxin concentrations and Q factor response, with OEU/ml displaying an increase in relative Q factor due to evaporation and IOEU/ml displaying a large decrease in Q factor in 80 seconds. At approximately 120 seconds the sensor response can distinguish between OEU/ml and 0.IEU/ml.

Figure 25 illustrates the relationship between relative 0-factor measured at 240 seconds, and the concentration of endotoxin in the standard being anaysed. It can be observed that biggest sensitivity to endotoxin is in the range of OEU/mI and 0.IEU/mI.

Figure 26 iDustrates the relationship between reaction chamber height and 0 factor response to viscosity/density standards in a sensor with 8.5mm long beams. It can be observed that if a sensor is desired with improved sensitivity to viscosity or density, the most optimal response (greatest change) is with a reaction chamber height (with the resonator at the centre) for this particular design is at around 1mm, However this device could use a reaction chamber height up to 1.4mm successfuUy. Longer beam devices wiD require a substantiafly greater reaction chamber, potentially a reaction chamber of several miflimetres to be optimally sensitive whilst a shorter beamed version would have an optimal response at a lower reaction chamber height.

Afl documents referred to in this specification are herein incorporated by reference. Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

The foHowing nonHmiting clauses are included as part of this specification: Clause 1. A method for the real time monitoring of the progress of a chemical reaction within a test sample by determining a change in the viscosity and/or density of the test sample, the method comprising the steps of: providing a fluid test sample, adding at least one reagent to the test sample to form a test sample mixture, immersing at least one resonant beam member in the test sample mixture, and determining a change in the viscosity and/or density of the test sample mixture by monitoring the change in at least one parameter relating to the resonance of the at least one resonating beam member wherein the parameter is selected from the group consisting of; resonance frequency, quality factor, and variation of the resonance phase angle of the at least one resonating beam member, and further using the change in said at least one parameter to determine the viscosity and/or density of the test sample mixture in order to determine the occurrence of a chemical reaction within the test sample mixture.

Clause 2. The method of clause 1 wherein the at least one beam member is part of a multi-beam resonator comprising 3 resonating beam members, each resonating beam members being fixed at either end of its longitudinal length and arranged in a substantially parallel arrangement to the other beam members.

Clause 3. The method of clause I or clause 2 wherein the method is used to determine the formation of a gel, coagulate, precipitate or agglutinate within the test sample mixture.

Clause 4. The method of any one of clauses I to 3 wherein the monitoring of the change in at least one of the parameters occurs repeatedly from a first time point when the at least one resonant beam member is immersed in the test sample mixture,to a second time point when a chemical reaction is identified as occurring within the test sample mixture.

Clause 5. The method of any preceding clause wherein at least one algorithm is used to process the data obtained relating to the change of at least one of the parameters in order to calculate an output measure relafing to the chemical reaction occurring in the test sample mixture.

Clause 6. The method of any preceding clause further comprising the step of calibrating the resonating frequency of the at least one resonating beam member prior to the at least one resonant beam becoming immersed in the test sample mixture.

Clause 7. The method of clause 6 wherein the calibration step comprises determining the environmental temperature in the area surrounding the multibeam resonator device.

Clause 8. The method of clause 6 wherein the calibration step comprises the step of determining the amount of protein present in a biological test sample, by determining the parameters relating to resonance frequency and quality factor upon immersion of the at least one resonating beam member in the test sample.

Clause 9. The method of any preceding clause wherein the test sample is a liquid selected from the group consisting of: a chemical sample, a biological sample, a pharmaceutical composition, a bodily fluid, blood, a blood product, and water or a water based product.

Clause 10. The method of clauses I to 9 wherein the chemical reaction which is monitored occurs during the performance of an immunoassay.

Clause 11. The method of clauses I to 9 wherein the chemical reaction results in the occurrence of agglutination within the test sample.

Clause 12. The method of any preceding clause wherein the at least one resonant beam member resonates at resonant mode I or resonant mode 3 or at a harmonic resonance.

Clause 13. A multi-beam resonator for use in monitoring a change in the density and/or viscosity of a test sample, comprising: -at least 3 resonant beam members, at east one vibratory element posiUoned to cause the vibration of at east one of the beam members.

at least one sensor means for determining at least one parameter associated with the vibration of the resonant beam member, -a reaction chamber defining a static voume, which is suitable for receiving and retaining a test sample, the reaction chamber comprising at east one inet port to aVow the introduction of the test sample and at east one ouflet port of dimensions which aVow for the outflow of air but not the test sample from the reaction chamber, wherein at east part of said at least one outet port has a hydrophobic surface coating.

Clause 14. The multi-beam resonator of clause 13 wherein the resonant beam members are provided in a paraflel arrangement and are fixed at each end of their longitudina' length to a base substrate.

Cause 15. The mu'ti-beam resonator of dause 13 or 14 wherein the at east in'et port aVows the test samp'e to be introduced into the reaction chamber from the top of the reaction chamber.

Cause 16. The mufti-beam resonator of any one of dauses 13 to 15 wherein at east part of the upper and bwer surfaces of the at least one ouflet port are coated with a hydrophHic material.

Clause 17. The multi-beam resonator of any one of clauses 13 to 15 wherein at east one outlet port is provided at at least one position seected from the group consisting of: above the reaction chamber, at a position which is radiafly furthest from the at east one inlet port, or in a location below the resonant assemby, wherein the at least one outlet port is provided in an arrangement and of dimensions which are suitable to permit air to escape.

Clause 18. The mufti-beam resonator of any one of clauses 13 to 17 wherein the at least one ouflet port has a depth of 9Opm and a width of around 0.6mm.

Clause 19. The multi-beam resonator of any one of clauses 13 to 18 wherein the ratio of the tota ongitudinal length of the resonant beam members to the length of the mounting zone of the muffi-beam resonator which is defined on the base substrate is in the range of I to 9.

Clause 20. The muffi-beam resonator of any one of clauses 13 to 18 wherein the ratio of the total longitudinal length of the resonant beam members to the length of the mounting zone of the multi-beam resonator which is defined on the base substrate is about 4.

Clause 21. The multi-beam resonator of any one of clauses 13 to 20 wherein the distance of the spacing between the outer lateral &de of the outermost resonant beam member and the innermost wall of the surrounding housing is 5mm or less.

Clause 22. The multi-beam resonator of any one of clauses 13 to 20 wherein the distance of the spacing between the outer lateral side of the outermost resonant beam member and the innermost wall of the surrounding housing is 0.5mm or less.

Clause 23. The multi-beam resonator of any one of clauses 13 to 21 wherein the distance of the spacing between the outer lateral side of the outermost resonant beam member and the innermost wall of the surrounding housing is greater than 2Spm.

Clause 24. The multi-beam resonator of any one of clauses 13 to 23 wherein the internal height of the reaction chamber is greater than 1 mm.

Clause 25. The multi-beam resonator of any one of clauses 13 to 24 wherein the width of the central resonating beam member in the lateral direction is about 2mm or less.

Clause 26. The multi-beam resonator of any one of clauses 13 to 24 wherein the width in the lateral direction of the central beam member is 1mm.

Clause 27. The multi-beam resonator of any one of clauses 13 to 26 wherein the multi-beam resonator comprises 3 resonating beam members and wherein the lateral width of the central beam member is the sum of the widths of the outer beam members.

Clause 28. The multi-beam resonator of any one of clauses 13 to 27 wherein the longitudinal length of the at least one resonant beam member is 18mm or less.

SI

Clause 29. The multi-beam resonator of any one of clauses 13 to 27 wherein the longitudinal length of the of the resonant beam members is about 5.5mm or less.

Clause 30. The muftibeam resonator of any one of clauses 13 to 29 wherein the distance of the spacing between resonant beam members which are arranged immediat&y paraflel to each other is 2mm or ess.

Clause 31. The multi-beam resonator of any one of clauses 13 to 30 wherein the total ongitudinal length of the multi-beam resonator is 20 mm or less.

Clause 32. The multi-beam resonator of any one of clauses 13 to 31 wherein each resonant beam member is capable of being resonated at a frequency of between about I kHz to about 500kHz.

Clause 33. The multi-beam resonator of any one of clauses 13 to 32 wherein the surface of at least part of the reaction chamber is provided with a hydrophobic coating.

Clause 34. The multi-beam resonator of any one of dauses 13 to 32 wherein the surface of at least part of the reaction chamber is provided with a hydrophilic coating.

Clause 35. The multi-beam resonator of any one of clauses 13 to 35 wherein the reaction chamber has an internal volume of less than 1000 microlitres, Clause 36. The multi-beam resonator of any one of clauses 13 to 35 wherein the reaction chamber has an internal volume of less than lOOpI.

Clause 37. The multi-beam resonator of any one of clauses 13 to 36 wherein the reaction chamber is comprised of a plurality of layers, wherein lower and upper layers define the base and lid of the reaction chamber respectively; and wherein these sayers are comprised from or coated with a hydrophilic material.

Clause 38. The multi-beam resonator of clause 37 wherein the reaction chamber further comprises a stainless steel layer from which the resonating beam members are defined.

Cause 39. The mut-beam resonator of any one of c'auses 13 to 38 wherein at east one surface of the reacton chamber is at east partiaUy coated a reagent which promotes the occurrence of a chemca reaction.

Cause 40. The mutibeam resonator of c'ause 39 wherein the reagent is amebocyte ysate or a synthetic ana'ogue thereof.

Ciause 41. A test strip comprising the mutibeam resonator of any one of dauses 14 to 40.

Cause 42. A method for determining the presence of endotoxin contamination within a fluid sampe by monitoring a change in the density and/or viscosity of the test samp'e, the method comprising the steps of: providing a test sampe, admxng the test sampe with a reagent comprising amebocyte ysate or a synthetic ana'ogue thereof in order to form a test samp'e mixture, immersing at east one resonant beam member of a mutibeam resonator device according to the invention in the test samp'e mixture, and determining a change in at east one parameter associated with the resonance of at east one resonating beam member seected from the group consisting of: (i) the resonance frequency of the at east one resonant beam member, (ii) the quafity factor of the at east one resonant beam member, (iii) changes in the resonance phase ange of the at east one resonant beam member r&ative to at east one further resonant beam, and using the observed change in said at east one parameter to cacuate the viscosity and/or density of the test sampe mixture, wherein an increase in the viscosity and/or density of the test sampe mixture is indicative of the presence of endatoxin within the test samp'e.

Cause 43. The method of dause 42, wherein in the amebocyte ysate is derived from Limuus amoebocyte ysate (LAL).

C'ause 44. The method of any one of cause 42 or 43 wherein the amebocyte ysate is provided in dried form.

Clause 45. The method of ciause 44 wherein the test sample is a fluid seected from the group consisting of: a pharmaceutica composition, a biologica' composition, a parenta preparation such as a diluent, carrier or adjuvant, a reconstitution buffer or salt solution for use aong with a pharmaceutica' composition, sterile water, natura water, purified water, treated water or distifled water.

C'ause 46. Use of a test strip of clause 41 for the continuous, reatime monitoring of the occurrence and progress of a chemica' reaction within a test sampe by monitoring a change in the viscosity and/or density of the test sampe wherein the chemical reaction results in the geilation, agglutination, precipitation or coagulation of the test sample.

Clause 47. Use of the method of any one of causes I to 12 for monitoring the progress of sri assay method or chemica reaction involving a change in the density and/or viscosity of a test sampe which is used for the assay method or chemical reaction.

Cause 48. Use of an apparatus of any one of dauses 13 to 40 for use in the monitoring of a test samp'e in order to determine a change in the viscosity and/or density of the test sampe, wherein the sampe is, or may be, undergoing a chemical reaction.

Clause 49. A kit for use in monitoring a change in the viscosity and/or density of a test sampe through the determination of at east one data value which is derived from a resonant beam member which is caused to resonate within the test sampe, said data parameter being used to calculate the density and/or viscosity of the test sampe in order to determine whether the test samp'e is undergoing a chemica reaction, the kit comprising a test strip according to the invention a'ong with instructions for the use of the same and the provision of appropriate reagents.

Cause 50. A test kit for detecting bacteria' endotoxin, said kit comprising: -a test strip comprising at east 3 resonant beam members which are provided within a reaction chamber having a defined interna voume and which is suitable to receive and retain a test sample, -a reagent comprising amebocyte ysate or a synthetic analogue thereof, -means for causing at east one of the resonant beam members to resonate and means to detect the resonation of at!east one of the resonant beam members, and -nstrucUons for the use of the same.

Claims (82)

1. Claims 1. Apparatus arranged for monitoring a change in the density and/or viscosity of a test sample, comprising: a multi-beam resonator comprising: a. a reaction chamber defining a volume, which is suitable for receiving and retaining a test sample, b. at least 2 resonant beam members within the reaction chamber, c. at least one vibratory element positioned to cause the vibration of at least one of the resonant beam members, d. at least one sensor means for determining at least one parameter associated with the vibration of the resonant beam member, e. the reaction chamber comprising at least one inlet port to allow theintroduction of a test sample,and further wherein a mounting zone of the multi-beam resonator is provided and the ratio of the total longitudinal length of the resonant beam members to the length of the mounting zone of the multi-beam resonator is in the range of I to 9.
2. 2. Apparatus according to claim I wherein the ratio of the longitudinal length of the resonant beam members to the length of the mounting zone of the multi-beam resonator is around 4.
3. 3. Apparatus according to claim 1 or 2 wherein the ratio of the longitudinal length of the resonant beam members to the length of the mounting zone of the multi-beam resonator is 1.59 or 3.67 or 4.06 or 4.36 or 8.72 or 9.33, or is between 1.59 and 4.67.
4. 4. Apparatus according to cLaim 3 in which the resonant beam members are 5.5mm in longitudinal beam length and the ratio of longitudinal beam length to the length of the mounting zone is between 1.59 and 4.67.
5. 5. Apparatus according to any preceding claim in which the total length of the multi-beam resonator is defined by the summation of the longitudinal length of the resonant beam member and the length of the mounting zone.
6. 6. Apparatus according to any preceding claim in which the length of the multi-beam resonator in the longest dimension is around 12.4mm.
7. 7. Apparatus according to any preceding claim in which an outer most portion of an outermost resonating beam is located a defined distance from walls which define the reaction chamber.
8. 8. Apparatus according to any preceding claim in which the resonating beams are arranged such that the distance between an inner surface of the reaction chamber and an outer periphery of the resonating beams minimises the shear effect between an inner surface of the reaction chamber and an outer periphery of the resonant beam members.
9. 9. Apparatus according to any preceding claim wherein the distance of the spacing between an outer lateral side of an outermost resonant beam member and an innermost wall of the surrounding reaction chamber is 5mm or less, or is 2mm or less, or is 0.5mm or less or is 0.5mm or greater or is approximately 200pm or is 100pm or is 20pm and/or is greater than 2Spm.
10. 10. Apparatus according to any preceding claim wherein the internal height of the reaction chamber is less than 1mm.
11. 11. Apparatus according to any preceding claim wherein the resonating beam members have a longitudinal length of 8.5mm and the height of the reaction chamber below the multi-beam resonator is greater than 0.1mm.
12. 12. Apparatus according to any of claims 1 to 10 wherein the resonating beam members have a longitudinal length of 5.5mm and the height of the reaction chamber below the multi-beam resonator is greater than 10pm, or wherein the resonating beam members have a longitudinal length of 5.5mm or less and the height of the reaction chamber is greater than 10pm, or wherein the resonating beam members have a longitudinal length of 14mm or less and the height of the reaction chamber between the multi-beam resonator and the base of the reaction chamber is 0.65mm.
13. 13. Apparatus according to any preceding claim wherein 3, 5, 7 or 9 resonating beam members are provided and the width of a central resonating beam member in the lateral direction is about 2mm or less.
14. 14. Apparatus according to claim 13 wherein the width in the lateral direction of the central beam member is 1mm.
15. 15. Apparatus according to any preceding claim wherein the multi-beam resonator comprises 3 resonating beam members and wherein a lateral width of the central beam member is the sum of the widths of the outer beam members.
16. 16. Apparatus according to any preceding claim wherein the longitudinal length of at least one resonant beam member is 18mm or less.
17. 17. Apparatus according to any preceding claim wherein the longitudinal length of the resonant beam members is about 5.5mm or less.
18. 18. Apparatus according to any preceding claim wherein the distance of the spacing between resonant beam members which are arranged immediately parallel to each other is 2mm or less or the distance of the spacing between 3 parallel resonant members is 0.75mm or less.
19. 19. Apparatus according to any preceding claim wherein the total longitudinal length of the multi-beam resonator is 20 mm or less.
20. 20. Apparatus according to any preceding claim wherein each resonant beam member is capable of being resonated at a frequency of between about I kHz to about 500kHz.
21. 21. Apparatus according to any preceding claim comprising a water proof layer.
22. 22. Apparatus according to claim 21 comprising a water proof layer of around 100pm.
23. 23. Apparatus according to claim 21 or 22 in which tracks and electrodes are provided and the apparatus further comprises a water proof layer covering the tracks and electrodes.
24. 24. Apparatus according to claim 21, 22 or 23 wherein a reaction chamber is formed around at least one resonant beam member by applying layers of water resistant material on both sides of the multi beam resonator and a substrate assembly.
25. 25. Apparatus according to any preceding claim in which at least 3, or 3, resonant beam members are provided.
26. 26. Apparatus according to any preceding claim in which the reaction chamber defines a static volume and allows a defined amount of test sample to be retained within the reaction chamber.
27. 27. Apparatus according to any preceding claim in which the reaction chamber includes an opening above the resonant beam members that is substantially open to the reaction chamber volume, permitting application of sample or escape of air from the reaction chamber.
28. 28. Apparatus according to any preceding claim in which a reaction chamber lid is provided above the resonating beam structures and an inlet port is provided within the reaction chamber lid.
29. 29. Apparatus according to any preceding claim in which an outlet port is provided to allow air to vacate the reaction chamber upon loading of the reaction chamber with test sample.
30. 30. Apparatus according to claim 29 in which at least part of the inner surface of the outlet port has a hydrophobic surface coating.
31. 31. Apparatus according to claim 29 or 30 in which the outlet port is located below the resonant beam members.
32. 32. Apparatus according to any preceding claim in which the multi-beam resonator is double ended so that the resonant beam members are fixed at each end of their longitudinal length.
33. 33. Apparatus according to any preceding claim in which the resonant beam members are parallel and fixed at each end of their longitudinal length to a base substrate.
34. 34. Apparatus according to claim 33 in which the resonant beam members are an integral part of the base substrate, or are an integral part of the base substrate and are formed by the technique selected from etching, photochemical etching, laser treatment and mechanical punching of the base substrate, or are an integral part of the base substrate and the reaction chamber comprises a stainless steel base substrate from which the resonating beam members are defined.
35. 35. Apparatus according to claim 33 in which the resonant beam members are not an integral part of the base substrate, or are not an integral part of the base substrate and are joined to the base substrate by a technique selected from: adhesive bonding, welding, mechanical assembly, soldering.
36. 36. Apparatus according to any of claims 33 to 35 in which the base substrate comprises one or more frame members parallel to the resonating beam members, or the base substrate comprises one or more frame members parallel to the resonating beam members and the one or more frame members are of differing length to the resonating beam members.
37. 37. Apparatus according to any preceding claim in which the resonant beam members are substantially composed of an inert material.
38. 38. Apparatus according to any preceding claim, in which the resonant beam members are composed of a material selected from the group of silicon, alumina, aluminium, copper, palladium, iron, gold, platinum, steel, stainless steel.
39. 39. Apparatus according to any preceding claim in which the vibratory element which mediates oscillation of the resonant beam members is a piezoelectric actuator.
40. 40. Apparatus according to any preceding claim in which the piezoelectric actuator comprises piezoelectric material and the piezoelectric material is selected from the group of PVDF (polyvinylidenedifluoride), crystal, ceramic, or the piezoelectric material is PZT (lead zirconate titanate) or the piezoelectric material is screen printed PZT (lead zirconate titanate).
41. 41. Apparatus according to any preceding claim in which the piezoelectric actuator is conjoined to a resonant beam member or is provided on the base substrate near the resonant beam member and electrical signal applied to the piezoelectric actuator results in vibration of the piezoelectric material forming the piezoelectric actuator and in turn vibration of the conjoined resonant beam member or of at least one resonant beam member located near the piezoelectric actuator when the piezoelectric actuator is provided on the base substrate.
42. 42. Apparatus according to any of claims 1 to 41 in which the vibratory element comprises magnetic shape memory materials or a transducer
43. 43. Apparatus according to any preceding claim in which the vibratory element is attached to at feast one resonant beam member and the sensor means is conjoined to a different resonant beam member, or; the vibratory element is attached to at least one resonant beam member and the sensor means is conjoined to the opposite end of the same resonant beam member, or; the vibratory element is conjoined to the base substrate and the sensor means is conjoined to a resonant beam member or to the base substrate in the proximity of the resonant beam members.
44. 44. Apparatus according to any preceding claim in which a plurality of sensor means are provided and are applied to different beam members or to the base substrate or to both the resonating beam members and to the base substrate.
45. 45. Apparatus according to any preceding claim in which the sensor means is a piezoelectric member or substantially comprised of piezoelectric material.
46. 46. Apparatus according to any preceding claim in which the vibratory element comprises a piezoelectric actuator, and the sensor means comprises a piezoelectric sensor, and electrical connections thereto are insulated from sample fluid in the reaction chamber.
47. 47. Apparatus according to any preceding claim in which sensor means for monitoring a parameter of at least one resonant beam member immersed in the test sample mixture comprises means for monitoring at least one parameter selected from the group of resonant frequency, quality factor, variation of resonance phase angle, change in resonant frequency, change in quality factor, change in resonant phase angle.
48. 48. Apparatus according to any preceding claim in which the sensor means for monitoring at least one parameter monitors the parameter relative to the same parameter as derived from one at least further resonant beam member also immersed in test sample.
49. 49. Apparatus according to any preceding claim in which the sensor means for monitoring a parameter comprises means for monitoring a change in a parameter selected from the group of resonant frequency, quality factor, variation in resonance phase angle continuously from a time point when the resonant beam member(s) is immersed in the test sample, or from a time point when the reagent is added to the test sample to form the test sample mixture.
50. 50. Apparatus according to any preceding claim in which the means for monitoring at least one parameter of at least one resonant beam member comprises means for indicating an increase in viscosity due to gellation of the test sample
51. 51. Apparatus according to any preceding claim for monitoring a change in the density and/or viscosity of the test sample, comprising: a. means for providing a test sample, b. means for admixing the test sample with a reagent in order to form a test sample mixture, c. means for immersing at least one resonant beam member of a multi-beam resonator device according to the invention in the test sample mixture, and d. means for determining a change in at least one parameter associated with the resonance of at least one resonating beam member selected from the group consisting of: i. the resonance frequency of the at least one resonant beam member, ii. the quality factor of the at least one resonant beam member, iii. changes in the resonance phase angle of the at least one resonant beam member relative to at least one further resonant beam, and e. means for using the observed change in said at least one parameter to calculate the viscosity and/or density of the test sample mixture.
52. 52. Apparatus according to any preceding claim in which the internal volume of reaction chamber volume is from about Sj.il to about 10d, is 35pl or is about 60tl or is less than lOOjil or is equal to or less than 1000jiI.
53. 53. Apparatus according to any preceding claim in which the change in the at least one parameter is monitored within 60s.
54. 54. Apparatus according to any preceding claimwherein the at least inlet port allows the test sample to be introduced into the reaction chamber from the top of the reaction chamber.
55. 55. Apparatus according to any preceding claim when dependent on claim 29 wherein at least part of the upper and lower surfaces of the at least one outlet port are coated with a hydrophilic material.
56. 56. Apparatus according to any preceding claim when dependent on claim 29 wherein at least one outlet port is provided at at least one position selected from the group consisting of: above the reaction chamber, at a position which is radially furthest from the at least one inlet port, or in a location below the multi-beam resonator, wherein the at least one outlet port is provided in an arrangement and of dimensions which are suitable to permit air to escape.
57. 57. Apparatus according to any preceding claim when dependent on claim 29 wherein the at least one outlet port has a depth of 9Oprn and a width of around 0.6mm.
58. 58. Apparatus according to any preceding claim wherein the surface of at least part of the reaction chamber is provided with a hydrophobic coating or comprises a material that displays hydrophobic properties or comprises a material modified to display hydrophobic properties or comprises material modified by plasma treatment to display hydrophobic properties.
59. 59. Apparatus according to any preceding claim wherein the surface of at least part of the reaction chamber is provided with a hydrophilic coating or comprises a material that displays hydrophilic properties or comprises a material modified to display hydrophilic properties or comprises material modified by plasma treatment to display hydrophilic properties.
60. 60. Apparatus according to any preceding claim wherein the reaction chamber is comprised of a plurality of layers, wherein lower and upper layers define the base and lid of the reaction chamber respectively, and wherein these layers are comprised from or coated with a hydrophilic material.
61. 61. Apparatus according to claim 60 wherein the reaction chamber further comprises a stainless steel layer from which the resonating beam members are defined.
62. 62. Apparatus according to any preceding claim in which a reagent is provided in dried form.
63. 63. Apparatus according to any preceding claim wherein at least one surface of the reaction chamber is at least partially coated with a reagent.
64. 64. A test strip comprising the apparatus of any one of claims 1 to 63 wherein a mounting zone of the multi-beam resonator is provided and the ratio of the total longitudinal length of the resonant beam members to the length of the mounting zone of the multi-beam resonator is in the range of 1 to 9.
65. 65. A method of monitoring a change in the density and/or viscosity of the test sample, the method comprising the steps of: a. providing apparatus comprising a multi-beam resonator assembly according to any of claims I to 63 or a test strip according to claim 64, wherein a mounting zone of the multi-beam resonator is provided and the ratio of the total longitudinal length of the resonant beam members to the length of the mounting zone of the multi-beam resonator is in the range of 1 to 9 b. providing a test sample, c. admixing the test sample with a reagent in order to form a teat sample mixture, d. immersing at least one resonant beam member of a multi-beam resonator device according to the invention in the test sample mixture, and e. determining a change in at least one parameter associated with the resonance of at least one resonating beam member selected from the group consisting of: (i) the resonance frequency of the at least one resonant beam member, (ii) the quality factor of the at least one resonant beam member, (iii) changes in the resonance phase angle of the at least one resonant beam member relative to at least one further resonant beam, and f. using the observed change in said at least one parameter to determine the viscosity and/or density of the test sample mixture.
66. 66. A method as claimed in claims 65 wherein at least one surface of the reaction chamber is at least partially coated with a reagent.
67. 67. A method as claimed in any of claims 65 to 66 wherein the test sample is a fluid selected from the group consisting of: a pharmaceutical composition, a biological composition, a parental preparation such as a diluent, carrier or adjuvant, a reconstitution buffer or salt solution for use along with a pharmaceutical composition, sterile water, natural water, purified water, treated water or distilled water.
68. 68. A method as claimed in any of claims 65 to 67 wherein the method is used to determine the formation of a gel, coagulate, precipitate or agglutinate within the test sample mixture.
69. 69. A method as claimed in any of claims 65 to 68 wherein the monitoring of the change in at least one of the parameters occurs repeatedly from a first time point when the at least one resonant beam member is immersed in the test sample mixture to a second time point when a chemical reaction is identified as occurring within the test sample mixture.
70. 70. A method as claimed in any of claims 65 to 69 wherein at least one algorithm is used to process the data obtained relating to the change of at least one of the parameters in order to calculate an output measure relating to the chemical reaction occurring in the test sample mixture.
71. 71. The method as claimed in any of claims 65 to 70 further comprising the step of calibrating the resonating frequency of at least one resonating beam member prior to the at least one resonant beam becoming immersed in the test sample mixture.
72. 72. The method as claimed in claim 71 wherein the calibration step includes determining the environmental temperature in the area surrounding the multi-beam resonator device, or further comprises resonating the resonating beams in air.
73. 73. The method as claimed in claim 71 wherein the calibration step further comprises the step of determining the amount of protein present in a biological test sample, by determining the parameters relating to resonance frequency and quality factor upon immersion of the at least one resonating beam member in the test sample.
74. 74. A method as claimed in any of claims 65 to 73 wherein the test sample is a liquid selected from the group consisting of: a chemical sample, a biological sample, a pharmaceutical composition, a bodily fluid, blood, a blood product, and water or a water based product.
75. 75. A method as claimed in any one of claims 65 to 74 wherein the chemical reaction which is monitored occurs during the performance of an immunoassay.
76. 76. A method as claimed in any one of claims 65 to 75 wherein the chemical reaction results in the occurrence of agglutination within the test sample.
77. 77. A method as claimed in any of claims 65 to 76 wherein the at least one resonant beam member resonates at resonant mode 1 or resonant mode 3 or at a harmonic resonance.
78. 78. Use of a test strip as claimed in claim 64 for the continuous, real time monitoring of the occurrence and progress of a chemical reaction within a test sample by monitoring a change in the viscosity and/or density of the test sample wherein the chemical reaction results in the gellation, agglutination, precipitation or coagulation of the test sample.
79. 79. Use of the method of any of claims 65 to 77 for monitoring the progress of an assay method or chemical reaction involving a change in the density and/or viscosity of a test sample which is used for the assay method or chemical reaction.
80. 80. Use of an apparatus as claimed in any of claims 1 to 63 for monitoring of a test sample in order to determine a change in the viscosity and/or density of the test sample, wherein the sample is, or may be, undergoing a chemical reaction.
81. 81. A kit arranged for monitoring a change in the viscosity and/or density of a test sample through the determination of at least one data value which is derived from a resonant beam member which is caused to resonate within the test sample, said data parameter being used to calculate the density and/or viscosity of the test sample in order to determine whether the test sample is undergoing a chemical reaction, the kit comprising: a test strip according claim 64 along with instructions for the use of the same.
82. 82. A test kit for according to claim 81, said kit comprising: -a test strip comprising at least 3 resonant beam members which are provided within a reaction chamber having a defined internal volume and which is suitable to receive and retain a test sample, -a reagent, -means for causing at least one of the resonant beam members to resonate and -means to detect the resonation of at least one of the resonant beam members, and -instructions for the use of the same.