WO2021191507A1 - Rotating solid support - Google Patents

Abstract

The invention relates to a method and apparatus for performing assays by using a rotating solid support.

Description

Rotating solid support

Background of the invention

A multitude of assay methods have been developed to determine analytes from samples. In determining and measuring different biomolecules from biological samples, immunoassays are often used, which play a particularly important role in analyzing, for example, patient samples. The already existing conventional assays, such as immunoassays carried out in microtiter plate wells, i.e. microwells, continuously need new improvements for the sensitivity, speed, specificity, costs, and performance of the assays. The assays are based on a specific binding of an analyte to a binder. In addition to the kinetics of the binding reaction, mass transfer in the proximity of the binding surface plays an important role in the speed of the assay - the faster the mass transfer and binding is, the shorter the total duration of the assay. Rapid mass transfer is necessary to minimise the local concentration reduction resulting from the binding of the analyte and the formation of an analyte concentration gradient in the proximity of the binding surface. If the rate of mass transfer is not sufficient to replace the bound analyte, the binding of the analyte to the binding surface slows down. Other steps of the assay include, for example, wash(es) to retain the binder-analyte complex formed by specific binding and to remove superfluous components. In particular, when using a labelled binder, the performance of the assay for measuring low analyte concentrations depends on how effective a solution exchange is achieved with the washes to remove the unbound labelled binder that is on the binding surface or that is weakly non- specifically bound on the binding surface, from the vicinity of the binding surface. A drying step(s) may also be carried out in the assay before the amount or concentration of the analyte is measured. Speeding up the washing and drying steps by optimizing their performance also shortens the time it takes to complete the entire assay. Optimisation of the assay binding, washing and drying steps may also play a role in the sensitivity of the assay, which can be affected e.g. by optimising the amount of specific binder-analyte binding.

The majority of assays in in vitro diagnostics are carried out in test systems where in a single assay the solid support is either a microwell or a group of microparticles. In this case, the binding reaction occurs in the area coated with binding molecules on the surface of the microwell or microparticles. These systems have been developed primarily for enzyme labels whose signal after the binding reaction and washes is formed as a result of an enzyme reaction in a solution from which it is measured either during or after the enzyme reaction. The signal to be measured is thus generated equally from the entire binding area of the solid support. On the other hand, there has not been much need for other types of measurements, as the normal fluorescence measured by surface measurement, for example, is not in practice suitable for sensitive immunoassays due to the background signal from the material of plastic solid supports. Assays carried out in microwells also show the problem that the excitation area used in direct fluorescence surface measurement at the bottom of the well is kinetically unadvantageous from the viewpoint of binding because binding in the conventional orbital shaking of the microwell takes place faster on the outer walls of the well and at the edges of the bottom than in the middle of the bottom, i.e. binding within the excitation area of the assay label measurement is slower than on the outside of the that area. Epifluorometric measurement of fluorescence from the excitation area at the bottom of the microwell plate is also a poor solution from the viewpoint of fluorescence measuring efficiency. Since fluorescence is typically evenly distributed in all directions, only a small portion of it can be collected from the solid angle limited by the high edges of the well and visible from the bottom through the well opening. In the case of microparticle solid supports, the binding is equal to the entire surface area of the microparticle group, but with fluorescence surface measurement it is practically impossible to measure the signal reproducibly from the entire particle surface area or even from a standard size fraction of the surface of the microparticle group. Conventional solid supports such as microwell plates and microparticles are therefore designed primarily for labelling techniques having a signal measured from a solution, and the labelling techniques based on surface measurement need a different solid support concept.

Brief description of the invention

It is therefore an object of the invention to develop a method and apparatus in order to solve the aforementioned problems. The object of the invention is achieved with a method and apparatus which are characterised by that which is disclosed in the independent claims. The different embodiments of the invention are disclosed in the dependent claims.

The invention relates to method for performing an assay, the method comprising steps of providing a rotating solid support which comprises a first binder, immersing the solid support in a solution which comprises an analyte, rotating the solid support to bind the analyte to the first binder, immersing the solid support in a wash solution, and rotating the solid support in the wash solution to improve the performance of the assay.

The invention is based on the use of a rotating body’s end surface that is immersed in liquid as a binding surface i.e. a solid support to which the binding molecules are attached and towards which a powerful liquid flow is directed as a result of a rotating movement. Rotation of the body is used both in the binding reaction and in the washing steps. It was surprisingly discovered that the use of rotation in addition to the analyte binding reaction also in the washing step(s) of the solid support improves the functioning of the assay. After the binding reaction, a signal may be measured as a surface measurement from the solid support, optionally while rotating it. Before measuring the signal, the solid support may be dried by, for example, rotating it. It was surprisingly discovered that drying by rotating improves the functioning of the assay.

The invention further relates to an apparatus for performing the assay, the apparatus comprising a rotating solid support, a first container comprising a solution comprising an analyte, and a washing container comprising a wash solution, whereby the solid support comprises a first binder, and whereby the apparatus is configured to immerse the solid support in the solution in the first container comprising an analyte, to rotate the solid support to bind the analyte to the first binder, to immerse the solid support in the wash solution in the washing container, and to rotate the solid support in the wash solution. The apparatus allows the inventive method to be carried out.

An advantage of the invention method and apparatus is the highly efficient mass transfer to the binding surface and, as a result of the binding of the analyte, minimisation of the concentration gradient of the analyte in the solution in the proximity of the binding surface. This effective mass transfer accelerates the implementation of binding-based bioaffinity assays and, in particular, improves the performance and sensitivity of rapid assays. Furthermore, the invention allows the binding surface to be efficiently washed and effective removal of unbound labelled binders. From the binding surface at the end of the body, the optical signal may be easily measured from a standard surface area and also collected efficiently without any obstacles to collection efficiency caused by the shape of the solid support, for example in fluorescence measurement. Brief description of the drawings

The invention will now be described in more detail in connection with preferred embodiments and with reference to the accompanying drawings, in which: Figure 1 shows an embodiment of the immunoassay of the invention.

(A) A bar-like solid support with a circular cross-section, an end of which is coated with an antibody, is immersed in a solution comprising an analyte so that at least the end of the bar is in contact with the liquid or below the liquid surface. During incubation, the solid support is rotated, whereby the rotation results in a hydrodynamic suction caused by centrifugal force, and the suction causes an effective fluid flow towards the binding surface, which minimises the formation of a concentration gradient in the proximity of the binding surface of the bar’s end as the analyte binds to the antibodies attached to the surface. (B) After analyte incubation, the solid support is transferred to the wash solution, where it is rotated and, among other things, the excess analyte is washed off. (C) Finally, the solid support may be dried by rotation, removing the liquid residues of the wash solution from the solid support surface.

Figure 2 shows the results obtained by surface measurement from a TSH immunoassay using so-called up-conversion luminescence, carried out on a rotating solid support surface. The assay is compared in Figure 2 with a conventional immunoassay carried out in a microwell with identical reagents and incubation times, where the signal was measured by surface measurement from the bottom of the 96-well plate. The luminescence (AU) is shown on the Y-axis, from which the background signal caused by the solid support material has been subtracted, and the X-axis shows the TSH content in nanograms per microlitre (ng/mΐ). Luminescence with an analyte concentration of 0 ng/mΐ is a background signal caused by non-specific binding to the solid support and it reflects the effectiveness of the washing step in removing labelled binders unbound to the analyte from the surface. The background signal in an assay performed with a rotating solid support is notably lower than in a conventional assay. Squares: conventional microwell assay; spheres: assay performed with a rotating solid support.

Figure 3 shows the results of an experiment that tested the binding of an analyte, i.e. a biotin-coupled europium chelate label (so-called bio-Eu-TEKES chelate), to a streptavidin-coated solid support using different rotation speeds, and comparing the result with a conventional microwell assay using a static (0 rpm) and mixed (950 rpm) assay. In the case of a bar-like solid support, the bar is rotated around its longitudinal axis, whereas in micro-pits the mixing takes place with orbital motion at the bottom plane of the microwell. The amount of analyte collected on the binding surface and thus the signal increased as the rotation speed of the solid support was increased. The signal increase caused by microwell shaking was notably lower. The Y-axis shows a surface-measured time-resolved fluorescence signal and the x-axis the rotation speed (rpm). Spheres: solid support assay, squares: microwell assay.

Figure 4 presents the results of an experiment to study the effect of rotation speed and mixing in an assay with a rotating solid support. The analyte used was bio-BSA-UCNP (UCNP coated with biotinylated BSA; UCNP, up- converted nanoparticle; BSA, bovine serum albumin). The binding speed was compared with the conventional assay performed in microwells. With a rotating solid support, the signal increased notably more than with a microwell, as the rotation speed was increased. The luminescence (AU) is shown on the Y-axis, from which the background signal caused by the solid support material has been subtracted, and the X-axis shows the rotation speed (rpm). Squares: solid support assay, spheres: microwell assay.

Figure 5 shows different options for patterning the solid support surface to enhance the suction flow rate and to increase the binding surface towards the cross sectional area of the bar.

Figure 6 shows an embodiment of the apparatus according to the invention. The method of the invention can be carried out with the apparatus. In the apparatus, the rotating solid support is transferred for different assay steps to, for example, the next liquid container or measuring step, In the exemplary assay, the apparatus carries out the binding step of the analyte to the binding surface, the washing step of the binding surface, the removal and drying of liquid residues from the binding surface, and, lastly, the measurement step.

Figure 7 shows the signal-background ratio (Sg/bg) in assays where washing has been performed statically (0 rpm) or by rotating the solid support at 1000 rpm. Sg/bg was considerably higher when washing was carried out by rotation compared to the static wash. Adding a second wash to the assay program increased the signal-background ratio compared with a single wash.

Figure 8 shows the background signal (Sg-bg) of the same assays for which signal-background ratios are shown in Figure 7. Sg-bg is calculated by subtracting from the signal of the control assay the signal caused by the solid support material. Sg-bg thus describes the non-specific binding of the label to the solid support. Washing by rotating significantly reduces this background signal compared to static washing. A plurality of washes reduces the background signal compared to one wash.

Figure 9 shows a background signal (Sg/bg) in assays where washing step has been performed statically (0 rpm) or by rotating the solid support at different speeds (50 to 6000 rpm). The results show that even the lowest rotation speed used, 50 rpm, lowered the average background signal compared to a static washing assay. The background signal was further reduced as the rotation speed was increased.

Detailed description of the invention

Current solid supports of assays, such as microparticles and microwells, have been developed to work primarily with enzyme labels, as their signal may be collected from the entire reactive surface area of the solid support into the solution by substrate incubation and measured from the solution. A similar method enabling signal collection from the entire surface area of a microwell plate by solution measurement is the measurement of a lanthanide label using DELF1A technology based on a separate development step. Surface measurement from the surface of the solid support, on the other hand, is, in principle, non-compatible with assays carried out in a microwell since, unlike in enzyme label assays containing a separate substrate incubation/development step, the surface measurement can read the signal from the bottom of the well for only a fraction of the total surface area of the solid support. Coating the well with a binding molecule may be limited only to the measuring area, but its location at the bottom of the well does not allow the diffusion layer to be effectively thinned at the binding surface by mixing the solution in the well/by shaking the well. However, surface measurement per se is a desirable feature of an assay, since no separate substrate incubation/development step is required after the binding reaction, but the binding reaction may be measured immediately after the washing step. This shortens the total time of the assay, which can be critical, for example, in point-of-care testing based on the use of the assay.

For surface measurement, the optimal solid support must enable thinning of the diffusion layer and an enhanced, even binding to the binding molecule-coated area combined with efficient washing and reproducible measurement of the area. In surface measurement, it would also be advantageous to be able to perform a measurement over the entire area coated with a binding molecule, as otherwise part of the binding will not be detected. In enzyme label assays containing the substrate/development step, the objective of effective and even binding has been achieved by means of a microparticle solid support, but surface measurement from microparticles is difficult to implement from a standard surface area, which reduces the reproducibility of the assay. Further, surface measurement of fluorescence labels requires that the shape of the solid support does not limit the effective collection of the signal from the binding surface, which is difficult to establish in the well plate assay due to the distance between the detector and the bottom of the well caused by the high walls of the well. In the case of a group of microparticles, it is possible to achieve a short distance between the detector and the microparticles, but a surface measurement cannot be carried out over the entire area because part of the surface area is always shaded on the back surfaces of the particles, and not reliably even in the standard part of the surface area, as particles may be grouped in several layers or at varying distances from each other in the excitation area, i.e. the number of particles to be measured varies between parallel reactions.

Surface measurement is therefore not optimally suitable for solid supports presently in use and it has not previously been considered as a starting point for the development of a new solid support concept. However, in particular if rapid assays are required, the use of surface measurement is desirable in order to minimise the total time of the assay. The present invention relates to a method for performing an assay, the method comprising providing a rotating solid support comprising a first binder, immersing the solid support in a solution comprising an analyte, rotating the solid support to bind the analyte to the first binder, immersing the solid support in a wash solution, and rotating the solid support in the wash solution to improve the performance of the assay. In the method, the assay of the analyte is performed by using, as a solid support, a rotating body suitable for surface measurements, which rotates in the direction of the longitudinal axis, the cross-section of which is substantially circular, and whose end surface at one end constitutes the actual solid support to which the binder is attached. The binding of the analyte to the binder is carried out by immersing the solid support in a solution containing an analyte, either by contacting the liquid surface or immersing below the liquid surface and by rotating the solid support, whereby with a rotating motion of the longitudinal axis a hydrodynamic suction is applied to the surface of the solid support, resulting in a strong liquid flow towards the surface. The solid support is typically immersed in the liquid perpendicularly to the liquid surface.

The term "solid support" may in this context refer to both a rotating elongated body and a surface at one end of it, that is, an end surface. This elongated body in question may also be described, for example, with the words drum, cylinder, rod, piston, bar, pillar or stick. The length of the elongated body is typically greater than the diameter of the cross section perpendicular to its longitudinal axis. The binder is implemented on the surface at one end of the body, that is, on the solid support. The surface may be a substantially flat surface. The surface may also be surface other than a substantially flat surface, whereby the surface shape allows for control of the hydrodynamic suction and liquid flow being generated. The surface shape may either be used to strengthen the hydrodynamic suction or to increase the area available to attaching the binder, thereby increasing the binder amount per the cross sectional area perpendicular to the rotation axis i.e. longitudinal axis of the body.

In an embodiment, the solid support is the surface at one end of the longitudinal body having a substantially circular cross section, and the body is rotated around its longitudinal axis.

In this context, the term "binder" refers to an analyte-binding molecule or structure which is used in an assay to identify the analyte and to bind to the analyte. A binder may be, for example, an antibody, an antibody fragment, a lectin, or an aptamer. In this context, the 'first binder' is the binder that is bound to the solid support. The binder may be attached to the solid support either covalently, passively by means of adsorption/chemsorption, or, for example, by means of a streptavidin-biotin interaction by using a streptavidin-coated solid support and a biotinylated binder. The analyte, in turn, attaches to the first binder. In this context, the 'second binder' is a binder that binds to the analyte attached to the first binder. The second binder is typically labelled with a label molecule that may be detected and measured quantitatively. Suitable label molecules and their measurement are well-known technologies for persons skilled in the art, which has surprisingly been found to be suitable for use in an assay carried out on a rotating solid support. What is particularly surprising is the advantages of employing different applications of surface measurement technology when using a rotating solid support. For example, the label may be a binder-coupled lanthanide chelate showing long lifetime luminescence after an excitation pulse or a nanoparticle emitting upconversion luminescence, the luminescence of which is measured by surface measurement on the surface of the solid support, either in a time-resolved manner after the excitation pulse or at the same time with excitation.

In this context, the term "analyte" refers to a molecule such as a biomolecule that binds to a binder or binders. The analyte may be a molecule in a sample, the amount or concentration of which in the sample is to be determined. Typically, the analyte may be a small molecule such as a hapten, peptide, protein, carbohydrate, nucleic acid or a complex or structure formed of one or more of these in different ways. Thus, the analyte may also be e.g. a glycoprotein, virus particle or part thereof, cell or external vesicle of the cell. For example, a sample may be a biological sample obtained from a subject such as a patient, such as a blood, serum, plasma, saliva, urine, faeces, tissue or spinal fluid sample. The sample may have been treated in any appropriate manner after sampling, such as dilution, centrifugation, filtration, precipitation, dialysis, chromatography, drying, reagent treatment, washing or enriching a specific component of the sample such as a cell population. The sample may also be a blood sample spot that is cut from a fibrous material on which the blood or other liquid sample has been dried. When using a blood spot sample, the rotating solid support allows for simpler assay automation than other types of solid supports, since the separation of the solid support and the blood spot is feasible in a simple manner and the blood spot sample hamper measuring the signal.

The analyte may also be present as a known quantity or concentration in a control sample, which is used, for example, as a control next to an unknown analyte content. The analyte may also be a labelled analyte, which is added to the sample in a known quantity. In this case, the labelled analyte competes with the (unlabelled) analyte in the sample for binding to the binder whereby the amount of analyte in the sample is found out by a so-called competing assay. In an embodiment, a solution comprising analyte additionally comprises labelled analyte.

In an embodiment, the method also comprises a step in which the solid support is immersed in a solution comprising a second binder, and a step in which the solid support is rotated to bind the second binder to the analyte bound to the first binder, whereby the second binder comprises a measurable label. The method may also include a second wash step in which the solid support is immersed in a second wash solution after the binding of the second binder, and the solid support is rotated in the second wash solution. In some embodiments of the method, the rotation may be continued or accelerated after washing in such a way that substantially all the wash solution can be removed from the solid support surface, and the solid support can be dried.

The hydrodynamic suction obtained by rotating the solid support causes in the sample solution a liquid flow that is perpendicular to the end surface of the solid support. The perpendicular flow results in a vortex-type liquid flow that is located below the surface of the solid support and is directed towards the surface, the flow continuously transporting the solution towards the centre of the surface and spreading laterally outward therefrom. The movement of liquid causes mass transfer of molecules in the proximity of the surface of the solid support, which is more efficient than a laminar liquid flow and the resulting mass transfer created in a well of a conventional microtitration plate by orbitally shaking the well and the liquid contained in it. More effective mass transfer reduces and eliminates local reduction in the analyte concentration resulting from the binding reaction and the formation of a concentration gradient close to the binding surface, which have an effect of limiting the reaction speed of the binding reaction. When the formation of a concentration gradient in the vicinity of the binding surface is prevented, the rate of the binding reaction is mainly only limited by the binding kinetics of the reaction. By speeding up the rotational motion, the binding reaction can also be accelerated at least up to a certain limit until the binding kinetics begin to limit the binding speed or the liquid flow generated by the rotational movement becomes turbulent. In the rotational movement, therefore, the binding to the surface in an optimal situation occurs as fast as physically possible with the analyte concentration in question. In the reaction accelerated by rotation, more analyte within the same time is bound to the same area than, for example, to the bottom of the well plate, and therefore the same sensitivity in the assay may be achieved faster, or when the same reaction time is maintained, a better assay sensitivity is achieved. Powerful mixing alone, and in particular combined with washes carried out with a rotational motion, also reduces non-specific binding, i.e. lowers the background signal of the assay, which also improves the sensitivity of the assay.

In the experiments presented herein, it has been shown that increasing the rotation speed of the solid support increases the binding rate of the analyte by enabling better mass transfer. The tests have also shown the effectiveness of the washing step carried out by rotating. In addition, the experiments presented herein have shown that the assay according to the invention achieves a better signal at all analyte concentrations than the reference assay performed in microwells, the non-specific background signal is lower and therefore sensitivity is better. For example, the sensitivity of a human thyrotropin or hTSH immunoassay in a conventional well assay was e.g. 13.17 ng/1, while in the assay carried out by the rotating solid support the sensitivity was significantly better, e.g. 0.95 ng/1.

The rotating solid support may be used in a heterogeneous assay in which the non-bound analyte and any other components of the sample are washed off the solid support after binding the analyte. Washing steps, which may include one or more washes, may also be carried out as a pre-wash before binding the first binder to the solid support or before binding the analyte to the first binder, or after binding the analyte before the binding step of the second binder. In this case, the solid support is immersed in a wash solution and the solid support is rotated to carry out the washing. The solid support is typically immersed in the liquid perpendicularly to the liquid surface. The washing step can be repeated several times by moving the rotating solid support to a new solution. The performance of a single washing step can be influenced by the volume of liquid in which the solid support is rotated. The solid support may, for example, be immersed deeper in solution during wash step(s) in order to improve the efficiency of washing than during label or analyte incubations. Alternatively or optionally, the solid support may be immersed in a series of several washing steps at different heights, for example by immersing the solid support deeper in a subsequent wash than in the previous wash.

As shown in the examples, the rotation speed used during the washing step affects the functionality of the assay, improving its performance. Compared to a static wash (rotation speed 0 rpm), a rotating wash increases the signal- background ratio and lowers the background signal. According to the results, the background signal also decreases as the rotation speed increases. A higher signal- background ratio from the same analyte concentration obtained by rotation during the washing step means a better sensitivity in the assay, because when a certain concentration differs more clearly from the background, it also allows for detection of lower concentrations than before, increasing the sensitivity of the assay. In other words, washing by rotation produces a significant advantage over static washing, improving or increasing the performance of the assay. In the invention, the solid support is rotated in the wash solution to increase (or improve) the performance of the assay, whereby the improvement of the performance becomes evident at least so that the assay may be implemented faster, whereby, optionally, the sensitivity stays at least the same, or an assay performed by rotating provides a better sensitivity, whereby, optionally, the duration of performing of the assay remains the same, or so that both of the above are realised.

The solid support may be dried between or after the different steps of the assay. Alternatively or optionally, the liquid can be removed by aspiration, i.e. suctioning it out. In this case, liquid residues may remain on the surface of the solid support. Drying can be carried out by rotating the solid support after it has been removed from the solution. In this case, the liquid on its surface is removed by centrifugal force and possibly also by airflow. Alternatively or optionally, drying can also be carried out using airflow, heat or warm airflow, for example. Alternatively or optionally, drying may be carried out using, for example, airflow, heat or warm airflow in combination with the rotation of the solid support.

In an embodiment a method, where the method additionally comprises one or more steps in which the solid support is dried. In another embodiment, the solid support is dried by rotation.

In an embodiment, the method comprises a step in which the amount of bound analyte is measured. The measurement is based on the amount of the label signal on the solid support whereby the label may be bound to a labelled analyte or another binder. The measurement is typically an optical measurement that can be performed, for example, as a colorimetric detection or luminescence detection, including photoluminescence, fluorescence, chemiluminescence, extended lifetime time-resolved fluorescence and detection of upconversion luminescence.

The rotating solid support of the present invention is a particularly optimal solid support for optical surface measurement, where a signal can be collected efficiently over the entire binding area. A solid support based on a rotating, binder-coated pillar end is a unique solution compared to existing commercial analyser systems because it utilizes hydrodynamic suction to the surface of the solid support, produced by the rotation movement, in the implementation of incubations and washings.

Upconverting nanoparticle labels enable a direct surface measurement, i.e. their luminescence may be measured on the surface of the solid support from the excitation area, unlike, for example, assaying of enzyme labels by chemiluminescence substrate incubation, which requires an additional incubation step. Avoiding an additional incubation step shortens the total time of the assay. Although the use of a rotating solid support pillar in immunoassays in connection with analyte binding has previously been presented in the literature (US 2008199880 Al; Driskell et al 2007, Surface-enhanced raman scattering immunoassays using a rotated capture surface. Anal. Chem. 79, 4141) its benefits for surface measurements have not been understood, as the surface measurement itself was not the starting point for the consideration. The rotating solid support pillar also enables in the assays enhanced washing steps by rotating the solid support also in the washing step and/or drying of the solid support by rotation. Optical surface measurement, especially by using upconversion luminescense, also enables free selection of solid support material, and the binding area attached to the end of the solid support need not be an absolutely even, separate gold surface, as in the solid support pillars previously described in the literature. When using upconversion luminescence and time-resolved fluorescence of lanthanide labels, the bar-like solid support and its end may be of the same material, allowing more cost-effective and simpler manufacturing. In other words, the inventive rotating solid support concept disclosed herein may bring a significant improvement in the performance of assays. In a fluorescence measurement, the rotating solid support also enables efficient and reproducible signal collection from the entire reaction surface, unlike well-like containers or the use of a group of microparticles. The rotating solid support can also be rotated during surface measurement, whereby possible variations in the excitation intensity or binding on the binding surface are averaged out.

Surface measurement may be carried out epifluorometrically from the solid support surface as a (photo)luminescence measurement. In this case, the pillar may be white or transparent (clear) material, e.g. polystyrene. Surface measurement can also be carried out using an optically transparent pillar as a fibre optic, in which case the solid support must be of a material that transmits both excitation light and emission light. A white solid support is particularly optimal for upconversion luminescence, as it enables particularly efficient collection of signal from the surface of the rotating solid support due to signal amplification and reflection.

An assay carried out with a rotating solid support is typically implemented in such a way that it is automated so that the solid support is transferred from one liquid well to another as shown in Figure 1. This method of implementation of the assay also allows several assays of different analytes to be made successively from the same sample by successively immersing a plurality of solid supports coated with different binders in the same sample. In this case, only a specific analyte is bound to each solid support from the sample, and another analyte may be measured thereafter. A limited amount of sample, from which only a few parameters could normally have been assayed, is sufficient for assaying several analytes. This is an advantage of the assay method of the invention compared to conventional assays, for example, in the assays carried out in microwells, a sample containing an analyte is washed out of the well after the analyte was bound, and only one analyte can be measured from the same sample.

In an embodiment, the solution comprising an analyte is a biological sample comprising several different analytes, and whereby two or more solid supports comprising different binders are sequentially immersed in the solution comprising analyte to determine a different analyte on each solid support.

In mixing, the centrifugal force weakens as the diameter of the solid support decreases. In an embodiment, the diameter of the solid support is less than 10 mm, preferably less than 6 mm, more preferably less than 4 mm. In an embodiment, the diameter is 4 mm. In another embodiment, the diameter is 4.5 mm. As the mixing motion decreases as the diameter of the solid support becomes smaller, the diameter of the solid support is preferably 0.5 mm, more preferably greater than 0.5 mm, even more preferably 1 mm, still more preferably larger than 1 mm. In an embodiment, the diameter of the solid support is 0.5 mm - less than 10 mm. In another embodiment, the diameter of the solid support is 0.5 mm - less than 6 mm. In yet another embodiment, the diameter of the solid support is between 0.5 mm and 4.5 mm. In yet another embodiment, the diameter of the solid support is 1 mm - 4 mm. In yet another embodiment, the diameter of the solid support is between 2 mm and 4.5 mm.

The size of the solid support also determines the sample and assay volume to be used. Typically, the sample volumes used are less than 100 mί, and by using a solid support with a diameter of more than 0,5 mm but no more than 10 mm in diameter, the best efficiency is achieved. When using a solid support of more than 10 mm diameter, the volume of assay solution and thus the sample volume should also be proportionally increased, or else the contact surface of the solid support per volume becomes too large, and the rotation motion of the entire solution increases at the expense of the hydrodynamic suction towards the solid support’s end. As the total available sample volume per single patient is limited, the sample volume used per analysis must also be limited. For example, when using in an assay a total volume of 50-250 mΐ that a sample forms either fully or of which the sample forms a part, e.g. 10 to 90 vol-%, the remaining part being buffer in which the sample is diluted, and when using a container which, as concerns its relevant dimensions, approximates a microwell of a C-type 96-well plate, i.e. the container has a substantially round cross section, its internal diameter is approximately 6 mm and the angle between the bottom and the inner wall is slightly diagonal, a solid support with a round cross-section and preferably having a diameter of 2 to 4.5 mm is suitable for the assay. In this case, the end of the solid support may either touch the liquid surface during the rotation motion or it may be between 0 mm and 4 mm below the liquid surface.

As shown in the experiments presented herein, the rotation speed of the solid support affects the binding rate of the analyte by enabling better mass transfer. Hydrodynamic suction was found to be enhanced, i.e. the functioning of the assay improved even at relatively slow rotation speeds. In an embodiment, the rotation speed is more than 0 rpm, preferably 300 rpm or more, more preferably 600 rpm or more, still more preferably 1000 rpm or more, even more preferably 1400 rpm or more. In another embodiment, the rotation speed is 300 rpm, preferably 600 rpm, more preferably 1000 rpm, even more preferably 1400 rpm. In another embodiment, the rotation speed is at most 6000 rpm, preferably at most 5000 rpm, more preferably at most 4800 rpm. In an embodiment, the rotation speed is more than 0 rpm to 6000 rpm. In another embodiment, the rotation speed is 150 to 6000 rpm. In yet another embodiment, the rotation speed is 300 to 4800 rpm. Further still, in an embodiment, the rotation speed is 300 to 1400 rpm. An advantageous rotation speed depends on the shape and diameter of the container, as well as on the diameter of the solid support. The rotation speed may be the same or different in different steps of the same assay.

In an embodiment, the rotation speed at the washing step or steps is 0 rpm, preferably 300 rpm or more, more preferably 600 rpm or more, still more preferably 1000 rpm or more, even more preferably at 1400 rpm or more. In another embodiment, the rotation speed is 300 rpm, preferably 600 rpm, more preferably 1000 rpm, even more preferably 1400 rpm. In another embodiment, the rotation speed is at most 6000 rpm, preferably at most 5000 rpm, more preferably at most 4800 rpm. In an embodiment, the rotation speed is more than 0 rpm to 6000 rpm. In another embodiment, the rotation speed is 150 to 6000 rpm. In yet another embodiment, the rotation speed is 300 to 4800 rpm. Further, in an embodiment, the rotation speed is 300 to 1400 rpm. The rotation speed may be the same or different in the different washing steps of the same assay.

The solid support pillar is immersed in different steps of the assay, i.e. in the binding of the analyte and label, and the wash(-es) under the liquid surface. How deep the surface of the solid support is immersed under the liquid surface depends, for example, on the shape and size of the container and volume of liquid used in the assay. However, the solid support surface at least touches the solution surface whereby it is 0 mm below the solution surface. In the washing step, the volume of wash solution is preferably as large as possible within the limits set by the volume of the container and rotation speed, because the larger the volume, the more efficient the washing will be. In an embodiment, the end of the solid support pillar is at least 1 mm below the liquid surface, preferably at least 2 mm, more preferably at least 3 mm, even more preferably 4 mm below the liquid surface.

In an embodiment, the solid support is raised above the liquid surface in its immediate vicinity after the rotation has started, for example to a height of not more than 1 mm. In this case, the hydrodynamic suction is maintained and the beneficial effects of the resulting fluid flow can be exploited without the sides of the rotating solid support being essentially in no contact with the liquid.

The solutions used in the assay, such as the sample, i.e. the solution containing the analyte, the wash solution, i.e. the wash buffer and the solution comprising the label, are in a container whose bottom typically has a round cross- section as seen from above. The inner surface of the bottom of the container is typically substantially flat, and the angle between the bottom of the container and the inner wall may be a right angle, rounded, bevelled or the bottom may be concave in its entirety (so-called U-shape). The diameter of the bottom of the container affects the volumes of liquid used in the assay, for example, so that the volume of liquid required to immerse the surface of the solid support is greater the larger the diameter of the bottom is. Typically, the assay is carried out by using volumes of liquid of less than 1 ml, e.g. 50 mΐ, 100 mΐ, 200 mΐ, 250 mΐ, 284 mΐ, 300 mΐ, 350 mΐ, 400 mΐ, 450 mΐ or 500 mΐ. The wash solution is typically used in greater volumes than analyte, binder or label solutions. The diameter of the bottom of the container is typically no more than 30 mm, preferably no more than 20 mm, more preferably about 10 mm, even more preferably about 6 mm.

The inventive solid support is typically an essentially flat surface. By patterning of the solid support surface, the mixing may be further enhanced even at lower rotation speed. In addition, patterning may be used to increase the reactive surface area of the solid support surface and the amount of binder molecules per cross-sectional area that binds to the surface. It is advantageous that the binding density on the solid support per cross-sectional area is as high as possible. The solid support surface may be embossed or inlaid either completely or at the outer edges so that the embossments or inlays of the pattern are radially from the centre of the pillar perpendicularly or diagonally to its outer edges. In addition, the patterning of the pillar end may be in accordance with the above, but such that the patterning or the plane surface in the middle is more elevated than the pattern at the edges of the pillar. Various options for patterning the solid support surface are shown in Figure 5. Inlays and/or embossing is harmful in a solid support located at the bottom of a conventional microwell, because in the laminar flow of mixing, a part of the bottom-located solid support is not within the flow and is therefore not in an advantageous position in the binding reaction or signal measurement. In the solid support of the invention, the fluid flow when rotating the solid support is perpendicular to the surface, which is beneficial for the flow of liquid on the patterned surface, provided that the embossment and/or inlays are such that the liquid flows in the patterned structure from the centre of the surface towards the edges. Therefore, the rotating solid support of the invention enables the utilisation of the embossed or inlaid solid support surface for increasing the amount of binders per solid support cross-sectional area and/or measuring surface, so that the entire amount of binders is also kinetically in an advantageous position unlike, for example, at the bottom of a microwell plate.

Alternatively or in addition to patterning, the cross-sectional area may be increased by coating the solid support surface with a polymer structure. Binder coating may be done as a monolayer on a flat or patterned solid support. Polymer coating allows a binder to be attached to a porous polymer layer on the solid support, such as in dextran chains, which allows a capacity greater than the monolayer. Both patterning and coating with polymer structure further improve the signal obtained with the assay of the invention over a certain period of time compared to the background signal.

In any embodiment, the binder may be attached to the solid support passively, covalently, or, for example, with a streptavidin-biotin bond. Alternatively or in addition to patterning and/or polymer coating, the end of the solid support may be treated with acid, gamma radiation, UV radiation or plasma or a combination thereof. The treatment enhances binding, such as the binding of streptavidin or the binding of an aptamer, lectin, antibody or antibody fragment, acting as the binder, to the solid support to coat it. It is preferable that the binding density on the solid support per cross-sectional area is as high as possible.

The material of the solid support may be plastic, such as polystyrene. Other examples of suitable solid support materials are glass, polyethene, polyvinyl chloride, nylon, acrylic such as polymethyl methacrylate, acrylonitrile butadiene styrene and polylactide. The end of the solid support may also have a porous or fibrous structure, such as porous glass or fiberglass. The solid support can also be an optically transparent pillar, allowing it to be used as fibre optic for measurement. In this case, the excitation light is supplied along the solid support, within it by making use of total reflection, to the end of the solid support, from which the excitation light is respectively collected along the solid support to a detector at the other end thereof. The solid support may also be optically opaque, e.g. white plastic, which further improves the collection efficiency of luminescence from the surface of the solid support, and fluorescence measurement may be made epifluorometrically from the end surface of a body. The solid support is typically disposable, whereby it is important that the solid support is simple and can be inexpensively manufactured and coated with a binding molecule. Recycling the solid support from one assay to the next requires full assurance that the performance of the binding surface will be maintained, but on the other hand, all the analytes collected in the previous assay have been removed.

The label used in the assay may be, for example, an enzyme label with a luminescent substrate, a luminescent or photoluminescent label. When using a luminescent substrate, the signal of the enzyme label is also formed at the location where the enzyme label is located, which corresponds to surface measurement. A photoluminescent label may be an upconverting nanoparticle or chelate, lanthanide chelate or optically fluorescent molecule. A luminescent label can be either a chemiluminescent or a fluorescent label that is electrochemically excited. Preferably the label is photoluminescent, such as an upconverting or extended lifetime lanthanide label. The photoluminescent label is measured by surface measurement directly from the solid support by applying an excitation light to the measuring area at the end of the rotating solid support. In surface measurement of a photoluminescent label, the solid support may be rotated during the measurement, whereby a representative, averaged result is obtained. When measuring upconversion luminescence, the solid support is, in an embodiment, a white plastic material such as polystyrene.

The invention also relates to an apparatus for performing the assay. The apparatus is able to carry out any embodiment of the assay of the invention and comprise any of the aforementioned features. An embodiment of said apparatus is shown in Figure 6 and it comprises a rotating solid support (1), a first container (2.1) comprising a solution (3.1) comprising an analyte (1.2) and a washing container (2.2) comprising a wash solution (3.2). The solid support comprises the first binder (1.1). As described in the above, the solid support (1) is a surface at one end of an elongated body, and the body is rotated around its longitudinal axis for example with the aid of an electric motor.

The apparatus is configured, e.g. by using an electric motor and a motion screw, to immerse the solid support (1) in a solution (3.1) comprising an analyte in the first container (2.1), to rotate the solid support to bind the analyte (1.2) to the first binder (1.1) to form a binder-analyte complex, to immerse the solid support in the wash solution (3.2) in the washing container (2.2), and to rotate the solid support in the wash solution. In practice, the apparatus used may comprise a control unit that includes a processor which, controlled by a computer program stored in the memory, controls the electrical motors included in the apparatus and other actuators needed to implement the necessary method steps.

The invention further relates to the use of said apparatus in the method.

There may be more than one washing container (2.2) in the apparatus, e.g. two or three or four containers, allowing more than one wash to be successively carried out for the solid support. Alternatively, the wash solution can be replaced in the washing container between washings. The apparatus may also have a separate pre-wash container to pre-wash the solid support before immersing it in a solution (3.1) containing an analyte.

The apparatus may be configured to dry the solid support. Drying may be carried out by rotating the solid support. In this case, the apparatus may comprise a drying container (2.3) in which the solid support (1) is dried by rotation and to which container the solution removed from the solid support accumulates. Alternatively or optionally, drying can also be carried out using airflow, heat or warm airflow, whereby the apparatus comprises means such as a blower or fan to generate airflow and/or means such as a heater to produce heat. Alternatively or optionally, the apparatus may carry out the drying by using, for example, airflow, heat or warm airflow in combination with the rotation of the solid support.

The apparatus may be configured to measure (4) the amount of analyte bound from the solid support (1). The measurement is based on the amount of the label on the solid support, which label may be bound to a labelled analyte or another binder. In such a case, the apparatus comprises means to carry out the measurement.

The apparatus may also comprise a second container comprising a solution comprising a second binder comprising the measurable label. In this case, the apparatus is configured to immerse the solid support in the solution in the second container comprising the second binder and to rotate the solid support in the solution comprising the second binder. The binding step of the second binder is carried out after the analyte is bound to the first binder to form a complex therebetween. The second binder binds to the analyte at a different site than the first binder, and forms a so-called sandwich, i.e. a first binder-analyte- second-binder complex. The second binder is typically labelled, whereby by measuring the label signal the amount of bound analyte may be assayed.

The apparatus may additionally comprise a second washing container comprising a second wash solution whereby the apparatus is configured to immerse the solid support in the second wash solution in the second washing container and to rotate the solid support in the second wash solution. The second wash in the second washing container is performed after the sandwich has been formed. There may be more than one second washing container in the apparatus, e.g. two or three or four containers, allowing more than one wash to be successively carried out for the solid support. Alternatively, the wash solution in the second washing container may be replaced between washings.

Alternatively or optionally, a solution comprising an analyte may also comprise a labelled analyte, in which case it is a so-called competing assay as described in the above. In this case, the signal of the label and the result of the assay is measurable without separate incubation with a second binder and the apparatus does not necessarily have to comprise a second container comprising a solution comprising a second binder and/or a second washing container.

In an embodiment, the bottoms of the apparatus's containers are substantially circular, i.e. the bottoms of the containers are round as seen from above. The inner surface of the bottom of the container is typically substantially flat, and the angle between the bottom of the container and the inner wall may be a right angle, rounded, bevelled or the bottom may be concave in its entirety (so- called U-shape).

The rotation speed of the solid support in the different containers 2.1, 2.2 and/or 2.3 may be 0 rpm, preferably 300 rpm or more, more preferably 600 rpm or more, still more preferably 1000 rpm or more, even more preferably 1400 rpm or more. In another embodiment, the rotation speed is 300 rpm, preferably 600 rpm, more preferably 1000 rpm, even more preferably 1400 rpm. In another embodiment, the rotation speed is at most 6000 rpm, preferably at most 5000 rpm, more preferably at most 4800 rpm. In an embodiment, the rotation speed is more than 0 rpm to 6000 rpm. In another embodiment, the rotation speed is 150 to 6000 rpm. In yet another embodiment, the rotation speed is 300 to 4800 rpm. Further, in an embodiment, the rotation speed is 300 to 1400 rpm. The rotation speed in the different containers of the same apparatus, such as in the container 2.1, 2.2 and/or 2.3 may be the same or different. An option to implement the apparatus is to utilize the apparatus sold under the brand name STEPCRAFT 2/D.3000 Construction Kit by STEPCRAFT GmbH & Co KG, An der Beile 2, 58708 Menden, Germany. In such a case, this apparatus may be programmed to implement the method steps carried out in the inventive method.

Examples

Example 1. Sensitivity ofTSH assay

The test determined a standard curve and sensitivity of a human thyrotropin (hTSH) immunoassay, performed by a rotating solid support and the result was compared to a conventional assay carried out in the micro wells of a 96-well plate. The rotating solid support was an end surface of a polystyrene bar, 4 mm in diameter, coated with streptavidin (SA) and polished. Polishing was carried out before the SA coating by polishing the bar, rotating its end with a grit 1000 sandpaper and then with a grit 2000 sandpaper. After SA coating, the solid support’s surface was blocked by incubation in bovine serum albumin (BSA) to reduce non-specific binding. The TSH-specific antibody 5409 was used as the label antibody in the assay, which had been labelled with an upconverting nanoparticle (UCNP) (5409-UCNP). The preparation of the antibody UNCP conjugates is described in the article Lahtinen et al. 2018 Mikrochim Acta. 13; 185. The binder, that is, capture antibody was a TSH-specific antibody 5404. Efforts were made to coat the capture antibody both on the end of the solid support and on the surface of the well plate at the same density, but since the surface area of the binding surface is larger in the microwell, the total amount of the antibody in the microwell is also higher than on the rotating solid support. The label antibody was used in solid support and microwell assays with substantially the same concentration and volume.

The reagents and means used in the assay were as follows:

• lx wash buffer (Wash buffer, Kaivogen Oy, Turku, Finland) and lx wash buffer + 1 mM potassium fluoride (KF)

• 20% polyacrylic acid (PAA), molecular weight (Mw) 1200, pH 7.5

• capture antibody: biotinylated anti-human-TSH monoclonal antibody (bio-Mab) 5404, concentration 378 ng/mΐ in a buffer 50 mM TSA pH 7.75 + 0.1 % BSA, biotinylation rate 78 % => biotinylated antibody 296 ng/mΐ

• Buffer Solution Colourless (42-03) (Kaivogen, Turku, Finland, Lot 1336)

• KGB + 7.5 % BSA, filtered with pore size with 0.22 pm filter (KGB = assay buffer, composition: 50 mmol/L Tris-HCl pH 7.75, 9 g/1 NaCl, 5 g/1 BSA, 0.1 g/1 Tween 40, 0.5 g/1 bovine gamma globulin, 20 mmol/1 DTPA and 0.5 g/1 NaNs)

• human TSH 1 mg/ml (> 90 % purity in buffer PBS + 0.1 % BSA + 0.1% NaN3, manufactured by Scripps laboratories, San Diego, USA, Lot 2582703), diluted in buffer KGB-BSA

• label antibody: anti-human-TSH UCNP-5409 in buffer KGB-BSA + 1 mM KF + 0.05 % (w/w) PAA

• wells for solid support assay in sample and label incubations: Nunc Modules 250 PP (ref 232034, Lot 111443, Thermo Scientific)

• wells for solid support assay in washing steps: polypropene F-wells (ref 655201, Lot E19843L9, Greiner Bio-one)

• microwells used in microwell assays SA96-C8 White SA-microtiter plate (Lot KG1216, Kaivogen Oy, Turku, Finland)

The 5409-UCNP dilutions containing PAA were prepared approximately 1.5 h before the start of the assays by adding reagents in the order KF, PAA, 5409-UCNP to the buffer. The solution was sonicated in an ultrasonic bath for 3 minutes just before use. The other dilutions were prepared and all solutions were pipetted on the plate just before the start of the assay. The buffers were tempered at room temperature for 30 minutes before the dilutions were prepared. The assay program also indicates the time spent for each step of assay and the relative height of the solid support in millimetres. The relative height was changed during the assay so that, for example, washings were carried out by immersing the solid support deeper in the solution than during antibody and analyte incubations. In this case, the washings were carried out in an enhanced manner. A step of each assay was carried out in its own well by moving the solid support from one well to the next and rotating the solid support in each well. The rotation speeds used are presented in the assay program. The Chameleon 10 microplate reader (Hidex, Turku, Finland) was used to measure the signal. TSH dilutions were used to obtain a standard curve at concentrations 0; 5.24; 0.328; 0.0205; 0.00128; 0.00008 and 0.000005 ng/mΐ. Three parallel assays were carried out with each different TSH concentration.

Assay program - solid support:

1. Pre-wash 7.5 s, 3500 rpm, 284 mΐ of wash buffer, -16.5 mm

2. Capture antibody bio-Mab 200 ng/100 mΐ, 4 min, 2000 rpm, 100 mΐ, -17.25 mm

3. Wash 7.5 s, 3500 rpm, 284 mΐ of wash buffer, -16 mm

4. TSH dilution 4 min, 2000 rpm, 100 mΐ, -17.25 mm

5. Wash 7.5 s, 3500 rpm, 284 mΐ of wash buffer, -16 mm

6. Label antibody 5409-UCNP 0.06 mg/ml, 4 min, 2000 rpm, 100 mΐ, - 17.25 mm

7. Wash 7.5 s, 3500 rpm, 284 mΐ of wash buffer, -15.5 mm

8. Wash 7.5 s, 3500 rpm, 284 mΐ of wash buffer, -16 mm

9. Wash 7.5 s, 3500 rpm, 284 mΐ of wash buffer + KF, -16,5 mm

10. Drying 5000 rpm 10s

11. Surface measurement from the solid support with Chameleon 10

Assay program - microwell:

1. Pre-wash of SA plate lx with plate washer

2. Add bio-Mab solution 50 mΐ/well

3. Incubation 4 min in a plate shaker, ss (= slow shaking), at room temperature (RT) 4. Wash xl with plate washer

5. Add TSH dilutions 50 mΐ/well

6. Incubation 16 min in a plate shaker, ss, RT

7. Wash xl

8. Add UCNP-5409 dilution 50 mΐ/well

9. Incubation 4 min in a plate shaker, ss, RT

10. Wash x4 with a plate washer, let dry under a paper 1 h

11. Surface measurement with Chameleon 10

The standard curves obtained from the various measurements are shown in Figure 2. A larger total amount of the capture antibody, or binder, was used in the microwell than on the rotating solid support because the surface area of the binding surface is larger on the well plate. In this case, the method using a rotating solid support has the advantage that by using a smaller binder amount the same or higher signal is achieved than in the conventional microwell assay. The upconversion luminescence is presented on the Y-axis as an arbitrary unit (AU), and on the x-axis the TSH concentration (mlU/1). The sensitivity in the conventional well assay was 13.17 ng/1, i.e. 0.142 mlU/1, while in assay carried out by the rotating solid support the sensitivity was significantly higher, 0.95 ng/1, i.e. 0.010 mlU/1. The sensitivity is determined as an analyte concentration corresponding to the background signal added by three times the standard deviation of the background. The assay with the rotating solid support did not have a measuring point below the sensitivity, so the result may not be accurate, but in any case the sensitivity of the assay with the rotating solid support is more than ten times better than that of the conventional microwell assay.

If the signals measured from the microwells are multiplied by a correction factor to correct the differences caused by the measurement height, the signals from the microwells and the rotating solid support are saturated at the same level. This reflects the fact that there are binders are present on the binding surface with the same surface density, and thus the same peak saturation density of analyte and labelled binder is achieved on both surfaces. Correction factor refers to a factor by which the signal measured from the bottom of the microwell has been corrected to correct the difference in collection efficiency compared to a signal measured from the end of the bar. The end of the bar can be measured closer to the detector and then the same label density at the end of the bar produces a higher signal than from the bottom of the well, the distance of which from the detector is limited by the height of the walls of the well. In other words, an equally large signal cannot be collected from the same surface density of labelled binder as from a rotating bar, which is one of the advantages of the method using a bar-like solid support compared to the microwell assay.

In Figure 2, the luminescence with an analyte concentration of 0 ng/mΐ is a background signal caused by non-specific binding to the solid support and reflects the effectiveness of the washing step in removing labelled binders unbound to the analyte from the surface. The background signal in an assay performed with a rotating solid support is notably lower than in a conventional assay, which means that rotation during a wash enhances the wash.

Example 2. Bio-Eu assay using different rotation speeds in the analyte binding step

In this test the binding of an analyte, i.e. a biotin-coupled europium chelate label (a so-called bio-Eu-TEKES chelate) to a streptavidin-coated (streptavidin = SA) solid support was tested using different rotation speeds in the binding step of the labelled analyte, and the result was compared with a conventional microwell assay performed in the wells of a 96-well plate, using both a static and mixed assay. The static assay did not use any orbital mixing, but the incubations were carried out without mixing. The solid support was the surface of the end of a polystyrene bar coated with streptavidin (SA), with a diameter of 4 mm, which had been polished before coating (polishing with 1000 + 2000 sanding paper by rotating the bar). After SA-coating, the solid support’s surface was blocked by incubation in bovine serum albumin (BSA) to reduce non specific binding.

The following materials and solutions were used in the test:

• bio-Eu-TEKES europium chelate (the chelate is structure 2 in publication Von Lode et al. 2003. A Europium Chelate for Quantitative Point-of-Care Immunoassays Using Direct Surface Measurement. Analytical Chemistry 75(13), 3193-3201), in HPLC eluent (20 mm TEAA, pH 7, dissolved in acetonitrile), concentration approximately 250 mM

• assay buffer: clear KGB buffer (Kaivogen, Turku, Finland: Lot KG1336)

• wash buffer: lx Wash buffer (Kaivogen, Turku, Finland)

• wells for solid support assay in sample and label incubations: Nunc Modules 250 PP (ref 232034, Lot 111443, Thermo Scientific) • wells for solid support assay in washing steps: polypropene F-wells (ref 655201, Lot E19843L9, Greiner Bio-one)

• microwells used in the microwell assay SA96-C8 White SA-microtiter plate (Lot KG1216, Kaivogen Oy, Turku, Finland)

The solutions were pipetted into the wells in the amounts according to the assay programs shown below. The assay program also indicated the time spent for each step of assay and the relative height of the solid support in millimetres. The relative height was changed during the assay so that, for example, washings were carried out by immersing the solid support deeper in the solution than during antibody and analyte incubations. In this case, the washings were carried out in an enhanced manner. The step of each solid support assay was carried out in its own well by moving the solid support from one well to the next and rotating the solid support in each well. The rotation speeds used are presented in the assay program. Three parallel assays were carried out with each different rotation speed. A Sense microplate reader (Hidex, Turku, Finland) was used to measure the signal. Three parallel assays were carried out with each different mixing speed.

Assay program - solid support:

1. Pre-wash of the solid support 7.5 s, 3500 rpm, 284 mΐ of wash buffer, -16.5 mm

2. bio-Eu-TEKES europium chelate solution 4 min, X rpm, 100 mΐ, - 17.25 mm a. X = 0 rpm b. X = 2400 rpm c. X = 4800 rpm

3. Wash 7.5 s, 3500 rpm, 284 mΐ of wash buffer, -15.5 mm

4. Wash 7.5 s, 3500 rpm, 284 mΐ of wash buffer, -16 mm

5. Wash 7.5 s, 3500 rpm, 284 mΐ of wash buffer, -16.5 mm

6. Wash 7.5 s, 3500 rpm, 284 mΐ of wash buffer, -16.5 mm

7. Drying 5000 rpm 10s

8. Surface measurement from the bar ends with the Sense reader.

The above assay performed with the solid support was compared with the conventional assay in SA-coated wells of a 96-well plate, where the bio-Eu chelate was bound to the inner surface of the well. The solutions used were the same as in the above. Two different mixings were used in the assay during the binding of the bio-Eu chelate - static (no rotation) or rotating mixing. Three parallel assays were carried out with each mixing type.

Assay program - solid support:

1. Wash lx with a plate washer

2. 100 mΐ of bio-Eu-TEKES dilution/well

3. Incubation 4 min in shaking (950 rpm) or stationary.

4. Wash x4 with a plate washer

5. Surface measurement from the wells with the Sense reader.

In this test, the binding of the analyte was tested with a small-sized analyte, i.e. the bio-Eu-TEKES chelate, and with different rotation speeds. In the conventional assay carried out in 96-well plate wells, static and mixed assay were used. The results (Figure 3) show that in the conventional assay, the use of mixing does not achieve a substantially better label binding to streptavidin, When using a solid support, the static (non-rotated) assay produced a weaker binding, i.e. a lower signal than the conventional assay, but instead the rotation speed of 2400 rpm of the solid support improved the binding kinetics, producing a significantly better binding, i.e. a higher signal, within the same binding time as the conventional assay. Raising the rotation speed of the solid support to 4800 rpm improved binding even more.

Example 3. Bio-BSA-UCNP assay

The experiment examined the effect of rotation speed and mixing in an assay with a rotating solid support. The analyte was bio-BSA-UCNP (BSA = bovine serum albumin), which is a larger molecule than the bio-Eu-TEKES chelate used in the above test. The binding speed was compared in the test with a conventional assay performed in the wells of a 96-well plate. The rotating solid support was the surface of the end of a polystyrene bar (diameter 4 mm) coated with streptavidin (SA), which had been polished before coating (polishing with 1000 + 2000 sanding paper by rotating the bar). After SA-coating, the solid support’s surface was blocked by incubation in bovine serum albumin (BSA) to reduce non-specific binding. The solutions were pipetted on the 96-well plate (conventional assay: SA96-C8 White, Streptavidin microtiter plate, Lot KG1216, Kaivogen Oy, Turku, Finland; rotating solid support assay: Nunc Modules 250 PP, ref 232034, Lot 111443, Thermo Scientific) in the amounts according to the assay programs shown below. The following solutions were prepared for the test:

• lx wash buffer (Kaivogen Oy, Turku, Finland)

• wash buffer (Kaivogen) + 1 mM KF

• Buffer Solution Colourless (42-03) (Kaivogen, Turku, Finland, Lot 1336)

• Label: UCNP-PAA-bio-BSA (6.63 mg/ml, in a KGB buffer)

• 20% polyacrylic acid (PAA), molecular weight (Mw) 1200, pH 7.5

• Biotin (Sigma), concentration 95 mM in buffer 250 mM NaOH, 9.5 mM Tris-HCl, pH 14,1; filtered with pore size 0.22 pm filter

The bio-BSA-UCNP dilutions containing PAA were prepared approximately 1.5 h before the start of the assays by adding the reagents in the order KF, PAA, bio-BSA-UCNP to the buffer. The bio-BSA-UCNP dilutions were sonicated in an ultrasonic bath for 3 minutes just before use. The other dilutions were prepared and all solutions were pipetted on the plate just before the start of the assay. The buffers were tempered at room temperature for 30 minutes before the dilutions was prepared. The assay program also indicated the time spent for each step of assay and the relative height of the solid support in millimetres. The relative height was changed during the assay so that, for example, washings were carried out by immersing the solid support deeper in the solution than during antibody and analyte incubations. In this case, the washings were carried out in an enhanced manner. Each step of an assay carried out with a solid support was performed in its own well by moving the solid support from one well to the next and rotating the solid support in each well. The rotation speeds used are presented in the assay program. The Chameleon microplate reader (Hidex, Turku, Finland) was used to measure the signal.

Assay program - solid support:

1. pre-wash 7.5 s, 3500 rpm, 284 mΐ of wash buffer, -16.5 mm

2. bio-BSA-UCNP 4 min, X rpm, 100 mΐ, -17.25 mm a. X = 150 rpm b. X = 300 rpm c. X = 600 rpm d. X = 1200 rpm

3. wash 7.5 s, 3500 rpm, 284 mΐ wash buffer + KF, -15.5 mm

4. wash 7.5 s, 3500 rpm, 284 mΐ wash buffer + KF, -16 mm 5. wash 7.5 s, 3500 rpm, 284 mΐ wash buffer + KF, -16.5 mm

6. drying 5000 rpm 10s

7. surface measurement from the bar ends with Chameleon 10

Assay program — microwell: the assay program was the same as the assay program described in the above in the test TSH assay sensitivity.

The results of the test are shown in Figure 4. Increasing the rotation speed increases the binding rate, allowing a better mass transfer. With a rotating solid support, the signal increased notably more than with a microwell as the rotation speed was increased.

Example 4. Effect of a wash carried out by rotating on the assay

In the experiment, the effect of a wash performed by rotating the solid support on the functioning of the assay was tested. The wash was performed statically by keeping the solid support in place (rotation speed 0 rpm) during the wash, or by rotation at the speed 1000 rpm. The solid support was an end surface of a polystyrene bar coated with streptavidin (SA).

The following materials and solutions were used in the test: · 5409-UCNP 50mM

• bio-RAM 0.261 mg/ml

• Kaivogen Buffer Solution Colourless (42-03), Lot KG1336

• PP-microplate U-form, Ref 650201, Lot B1901337, Greiner Bio- one · PP-MICROPLATE, F-bottom (flat), Ref 655201, Greiner Bio-one

• l x wash buffer, Kaivogen

• l x wash buffer, Kaivogen + 1 mM KF

TSH-specific antibody 5409 was used as the label antibody in the assay, which had been labelled with an upconversion nanoparticle (5408-UCNP). The capture antibody was bio-RAM i.e. biotinylated rabbit anti-mouse antibody. The solutions were pipetted into the wells in the amounts according to the assay program shown below. The assay program also indicated the time spent for each step of assay and the relative height of the solid support in millimetres. The relative height was changed during the assay so that, for example, washings were carried out by immersing the solid support deeper in the solution than during antibody and label incubations. In this case, the washings were carried out in an enhanced manner. The second washing was optional, so it is marked with parentheses in the assay program. In the second wash, potassium fluoride (KF) was used in the buffer to prevent erosion of UCNP. Each step of an assay carried out with a solid support was performed in its own well by moving the solid support from one well to the next and rotating the solid support in each well. During the washing step (washing steps), F- i.e. flat-bottomed microplate wells were used, and in the other steps U-shaped wells were used to minimise the solution volume. The rotation speeds used are presented in the assay program. The Chameleon microplate reader (Hidex, Turku, Finland) was used to measure the signal.

Assay program:

1. Pre-wash 7.5 s, 3500 rpm, 284 mΐ of wash buffer, -16.5 mm

2. bio-RAM (0.05 ng/mΐ tai 0 ng/mΐ) 15 min, 500 rpm, 170 mΐ, -17.6 mm

3. Wash 7.5 s, 3500 rpm, 284 mΐ of wash buffer, -16 mm

4. TSH-UCNP (0.004 mg/ml) 4 min, 2000 rpm, 170 mΐ, -17.6 mm

5. Wash 7.5 s, 0 rpm / 1000 rpm, 284 mΐ of wash buffer, -16.5 mm

(6. Wash 7.5 s, 0 rpm / 1000 rpm, 284 mΐ of wash buffer, -16.5 mm)

The results are shown in Figures 7 and 8. Figure 7 shows the signal- background ratio (Sg/bg) in assays where washing has been performed by rotating at the speed of 1000 rpm or by keeping the solid support in place (0 rpm). Sg is a signal obtained with the capture antibody concentration of 0.05 ng/mΐ, and bg is a signal obtained without capture antibody. From both signals Sg and bg, the signal measured from the SA-coated solid support without the label and capture antibody was first subtracted. Sg/bg was considerably higher when washing was carried out by rotation compared to the static wash. Adding a second wash to the assay program increased the signal-background ratio compared to a single wash. Figure 8 shows the background signal (Sg-bg) obtained from the same assays, wherein a signal measured from the SA-coated solid support without a label and capture antibody has been subtracted from the signal of the control assay carried out without the capture antibody bio-RAM. Sg-bg thus denotes the non-specific binding of the label 5409-UCNP to the solid support. Washing reduces the background signal significantly better when the wash is carried out by rotating and not by keeping the solid support in place. A plurality of washings reduces the background signal compared to one wash.

Example 5. Effect of washing with different rotation speeds

In this test the washing of the solid was tested at different rotation speed of 0 rpm, 50 rpm, 100 rpm, 500 rpm, 2000 rpm and 6000 rpm. In other respects, the assay was performed in the same way as in example 4 above. Only one wash was performed, so the optional wash step of the assay program was not carried out.

Figure 9 shows the results of the experiment, i.e. the signal- background ratio (Sg-bg) in the assays where the washing step was performed by rotating at different speeds between 50 and 6000 rpm or by keeping the solid support stationary (0 rpm). The results show that even the lowest rotation speed used, 50 rpm, lowered the average background signal compared to an assay using static washing. The background signal was further reduced as the rotation speed was increased. The signal-background ratio Sg/bg (results not shown) was also higher at all the different rotation speeds than in the assays with a static wash.

Those skilled in the art will find it obvious that, as technology advances, the basic idea of the invention may be implemented in many different ways. The invention and its embodiments are thus not restricted to the examples described above but may vary within the scope of the claims.

Claims

Claims

1. A method for performing an assay, said method comprising the following steps: providing a rotating solid support comprising a first binder, immersing the solid support in a solution comprising an analyte, rotating the solid support to bind the analyte to the first binder, immersing the solid support in a wash solution, and rotating the solid support in the wash solution to improve performance of the assay.

2. The method as claimed in claim 1, wherein the method further comprises a step of immersing the solid support in a solution comprising a second binder, and a step of rotating the solid support to bind the second binder to the analyte bound to the first binder, wherein the second binder comprises a measurable label.

3. The method as claimed in claim 2, wherein the method further comprises a second washing step of immersing the solid support in a second wash solution after the binding of the second binder, and rotating the solid support in the second wash solution.

4. The method as claimed in claim 1, wherein the solution comprising an analyte additionally comprises a labelled analyte.

5. The method as claimed in any one of the preceding claims, wherein the method additionally comprises one or more steps of drying the solid support.

6. The method as claimed in any one of the preceding claims, wherein the solid support is dried by rotating.

7. The method as claimed in any one of the preceding claims, wherein the method additionally comprises a step of measuring the amount of bound analyte.

8. The method as claimed in any one of the preceding claims, wherein the solid support is a surface at one end of an elongated body, and the body is rotated about its longitudinal axis.

9. The method as claimed in any one of the preceding claims, wherein the solid support has a diameter of 0.5 mm to less than 10 mm.

10. The method as claimed in any one of the preceding claims, wherein the rotation speed is more than 0 rpm, optionally wherein the rotation speed in the washing step is more than 0 rpm.

11. The method as claimed in any one of the preceding claims, wherein the solid support is immersed in the solutions at least 3 mm below the liquid surface.

12. The method as claimed in claim 11, wherein the solid support is raised no more than 1 mm above the surface of the liquid after starting rotation.

13. The method as claimed in any one of the preceding claims, wherein the solution comprising an analyte comprises a multitude of different analytes, and wherein two or more solid supports comprising different binders are sequentially immersed in the solution comprising an analyte to assay a different analyte with each solid support.

14. The method as claimed in any one of the preceding claims, wherein the solid support is plastic, preferably polystyrene.

15. An apparatus for performing the method as claimed in any one of claims 1 to 14, said apparatus comprising a rotating solid support, a first container comprising a solution comprising an analyte, a washing container comprising a wash solution, wherein the solid support comprises a first binder and wherein the apparatus is configured to: immerse the solid support in the solution comprising an analyte in the first container, rotate the solid support to bind the analyte to the first binder, immerse the solid support in a wash solution in a washing container, and rotate the solid support in the wash solution.

16. The apparatus as claimed in claim 15, the apparatus further comprising: a second container comprising a solution comprising a second binder comprising a measurable label, wherein the apparatus is configured to: immerse the solid support in the solution comprising a second binder in the second container, rotate the solid support in the solution comprising a second binder.

17. The apparatus as claimed in claim 16, the apparatus further comprising a second washing container comprising a second wash solution, whereby the apparatus is configured to: immerse the solid support in the second wash solution in the second washing container, and rotate the solid support in the second wash solution.

18. The apparatus as claimed in claim 15, wherein the solution comprising an analyte additionally comprises a labelled analyte.

19. The apparatus as claimed in any one of the preceding claims 15 to 18, wherein the apparatus is configured to dry the solid support, optionally wherein the drying is carried out by rotating.

20. The apparatus as claimed in any one of the preceding claims 15 to 19, wherein the apparatus is configured to measure the amount of bound analyte.

21. The apparatus as claimed in any one of the preceding claims 15 to 20, wherein the solid support is a surface at one end of an elongated body, and the body is rotated about its longitudinal axis.

22. The apparatus as claimed in any one of the preceding claims 15 to 21, wherein the shape of the bottom of the containers is substantially round.

23. Use of the apparatus as claimed in any one of the preceding claims 15 to 22 in the method as claimed in any one of claims 1 to 14.