WO1992010743A1

WO1992010743A1 - Method of determining a specific ligand in a liquid sample with the aid of an acoustic measuring device, and a component of the measuring device

Info

Publication number: WO1992010743A1
Application number: PCT/NL1991/000258
Authority: WO
Inventors: Richardus Bernardus Maria Schasfoort; Maarten Simon Nieuwenhuizen
Original assignee: Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek TNO
Current assignee: Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek TNO
Priority date: 1990-12-11
Filing date: 1991-12-10
Publication date: 1992-06-25
Anticipated expiration: 1993-06-11
Also published as: NL9002724A

Abstract

The invention relates to a method of determining a ligand in a liquid sample by bringing the sample into contact with a vibrating object which is accommodated in an acoustic measuring device and whose surface is provided with specific binding partners for the ligand to be determined, and analysing the frequency shift or the change in the acoustic propagation velocity or the change in the amplitude or the phase change measured at the vibrating object and due to the formation of a complex between the ligand and the specific binding partner, which method is characterised in that a vibrating object is used whose surface situated at the liquid/solid interface is provided with either a multiplicity of latex spherules having a diameter of 50-1000 nm as particulate carriers which contains specific binding partners for the ligand; or a multiplicity of specific binding partners which are provided with a 'leaving agent' in the form of a particle reversibly bound to said specific binding partners and being a ligand on a latex spherule having a diameter of 50-1000 nm; or a multiplicity of specific binding partners for the ligand which are partly provided with the ligand and the 'leaving agent' in the form of a ligand on a latex spherule having a diameter of 50-1000 nm, which ligand is reversibly bound to said specific binding partners in accordance with an established equilibrium with a medium (competition reaction) which contains the ligand and the 'leaving agent', said established equilibrium being temporarily shifted after introducing a sample to be examined resulting in a corresponding frequency shift.

Description

Method of determining a specific ligand in a liquid sample with the aid of an acoustic measuring device, and a component of the measuring device.

The invention relates to a method of determining a specific ligand in a liquid sample by bringing the sample into contact with a vibrating object which is accommodated in an acoustic measuring device and whose surface is provided with specific binding partners for the ligand to be determined, and analysing the frequency shift or the change in the acoustic propagation velocity or the change in the amplitude or the phase shift measured at the vibrating object and due to the formation of a complex between the ligand and the specific binding partner.

Such a method is disclosed by Thomson, M., et al., "The Potential of the Bulk Acoustic Wave Device as a Liquid-Phase Immunosensor", IEEE Transactions on Ultrasonics, Ferroelectries and Frequency Control, Vol. UFFC-3*}, No. 2, March 1987. This literature reference deals in detail with the interface reactions in aqueous media of, inter alia, antibodies with antigens immobilised at the surface of the measuring crystal used in the BAW sensor (see Figure 1). More generally, it can be stated that, in a gaseous medium, the change in frequency of a vibrating quartz crystal due to adsorbed mass can be precisely related to said adsorbed mass using the Sauerbrey theory (see Czanderna and C. Lu (1984), Applications of Piezoelectric Quartz Crystal Microbalances, Methods and Phenomena, Vol. 7, Elsevier, Amsterdam, in particular, page 31): t\f = S.Δm(l) S = -2₀ ²/Z_q(2) where t\f - the frequency shift with respect to „ ₀ = the unloaded resonance frequency of the crystal Am = the adsorbed mass Z_q = the shear impedance of quartz (p_q.V_q = density of quartz x transverse wave velocity in quartz). For a 20 MHz AT-cut quartz crystal (resonating at 20 MHz), for which p_q = 2650 kg/m³ and V_q = 3340 m/s (see also page 31 of Czanderna and Lu, loc.cit.), the mass sensitivity S measured in the gas phase is approximately 1 Hz/(ng/cm²), in other words, the adsorption of 1 ng of mass/cm² of surface of the quartz crystal, measured in the gas phase, results in a frequency shift of 1 Hz.

It can also be inferred from the abovementioned literature reference of Thompson, M. et al. (loc.cit.) that the quartz crystals are severely damped in liquid, and this results in an increase in the noise and the "drift" of the signals obtained from the measuring crystal, with the result that the minimum detection limit of the BAW measuring device is adversely affected.

With respect to EP-A- 0.215-669, page 4, line 39 - page 6, line 13, in particular page 4, lines 9~ 4 and Example 4, first ^" paragraph, it is remarked that according to said EP-A- the sensitivity of the piezoelectric crystal biosensor may be enhanced if, after samples have been exposed to the surfaces of the piezoelectric crystal, latices or other fine particles on which an antibody or an antigen is immobilized, are passed over these surfaces and are bound by means of antigen-antibody reaction via the attached ligand to the surface of the piezoelectric crystal. In this way the weight change of the surface of the piezoelectric crystal is amplified with said latices or other fine particles. However, concerning the contents of these passages in EP-A- 0.215-669 it is brought to the fore that relatively high demands have to be made with respect to the quality of the layer of antibodies or antigens, which is applied to the surface of the piezoelectric crystal in order to be able to obtain measurements having sufficient sensitivity.

Concerning W0 89/09937 it is brought to the fore, that said reference discloses a method, which encompasses the use of a conjugate, comprising an enzyme, which is coupled to either (a) the analyte or (b) an anti-analyte-reagent. For that reason the conjugate is able to bind itself to either (a) the "capture" reagent bound to the surface of the quartz crystal or (b) the analyte bound to said "capture" reagent. Then a substrate is added to this system which substrate is specific for the enzyme and is converted under the influence of this enzyme (see page 5t lines 12-19 of W0 89/09937)- However, the use of such an enzyme-containing conjugate and the respective substrate which is specific for the enzyme is considered laborious and most likely the enzymatic conversion in question merely gives a qualitative measurement of the analyte present in the unknown sample.

In view of WO 89/09938, in particular the passage on page 5. lines 3~10, it is brought up that the measurement is based on competition, according to which the analyte is bound to either the binding agent on the modified "massive" sol particle of for instance Au or to the binding agent on the modified quartz crystal of the measuring device, whereby in the case of a binding between analyte and sol particle, the sol particle in question cannot stick any more to the surface of the quartz crystal and as the consequence thereof a response is measurable in comparison to the case wherein no analyte is present. However, according to this embodiment the sol particles loaded with analyte should remain in solution and may not precipitate on the surface of the quartz crystal. A principal different approach is based on the embodiment as described on page 5, lines 11-15 of WO 89/09938 wherein the modified sol particle containing several receptors for the analyte may be bound to the analyte bound on the surface of the quartz crystal. In case of a sufficient analyte concentration all receptor sites of the sol particle are occupied, so no binding is possible anymore between the massive sol particles and the quartz crystal surface (see Fig. 1 of W0 89/09938) . However, the use of such sol particles comprising several receptors for the analyte make this method rather laborious and little sensitive as

- as indicated in Fig. 1 of W0 89/09938 - there will be hardly any difference between the occupation of none, one, two or three receptor sites of a "massive" sol particle which has bound itself with the fourth receptor site to the quartz crystal surface. Only in the case of the occupation of also the fourth receptor site by an analyte a clear frequency shift will occur. Therefore, such a measurement merely indicates the presence of either an excess of analyte or the absence or the presence of a "small" amount of analyte, so this method seems to be fit for qualitative and not or hardly for quantitative measurements. In view of the need for a fast and sensitive method of carrying out, inter alia, immunoassays, the Applicant has sought a methodology which can eliminate the disadvantages, such as the undesirably high detection limit, of acoustic methods of measurement, such as the "bulk acoustic wave" method described above and also other acoustic methods of measurement such as the "plate wave" technique, occurring in liquid.

Surprisingly, it has been found that the abovementioned object can be achieved if, in the method described at the outset, a vibrating object is used whose surface situated at the liquid/solid interface is provided with a multiplicity of latex spherules having a diameter of 50-1000 nm as particulate carriers which contain specific binding partners for the ligand (see Figure 2). Contrary to the above discussed methodology according to EP-A- 0.215-669 it is remarked that according to the invention a multiplicity of particulate latex carriers is applied directly to the surface of the vibrating object present in the acoustic measuring device which latex particles comprise several binding partners specific for the ligand. For that reason the above-set demand for the EP-A- 0.215-669 is less relevant for the invention, i.e. the sensitivity of the surface of vibrating object, build up according to the invention is already high on its own. Moreover, many degrees of freedom and coupling possibilities of the affinity ligands, as for instance illustrated in Fig. 5_t are possible, which aspect is not mentioned or suggested in EP-A- 0.215.669.

The abovementioned object can also be achieved with the aid of a vibrating object whose surface situated at the liquid/solid interface contains a multiplicity of specific binding partners which are provided with a "leaving agent" in the form of a particle reversibly bound to said specific binding partners being a ligand on a latex spherule having a diameter of 50-1000 nm (see Figure 3) • According to an embodiment of the invention the specific binding partners for the ligand may also be bound to a multiplicity of latex spherules having a diameter of 50-1000 nm as particulate carriers, which spherules are attached to the surface of the vibrating object. The abovementioned object can also be achieved with the aid of a vibrating object whose surface situated at the liquid/solid interface contains a multiplicity of specific binding partners for the ligand which are partly provided with the ligand and the leaving agent in the form of a ligand on a latex spherule having a diameter of 50-1000 nm which is reversibly bound to said specific binding partners in accordance with an established equilibrium with a medium (competition reaction) which is advantageously circulated and which contains a constant concentration of the ligand and the "leaving agent", said established equilibrium being temporarily shifted, resulting in a corresponding frequency shift, after introducing a sample to be investigated (Figure 4a) . In this respect it is pointed yet at page 9. second paragraph and Fig. 2 respectively of WO 89/09938 showing a competition reaction between on the one hand an analyte and on the other hand "massive" sol particles of for instance Au or Pd, which are provided with one or more analytes. However, it is not indicated at all in this passage that latex particles having a diameter of 50-1000 nm and provided with one or more analytes, may be used in a competative measuring methodology according to which these latex particles are in equilibrium with the analyte and - after introduction of a sample to be examined - this equilibrium may temporarily shift and yield a corresponding frequence shift.

An important advantage of this last-mentioned achievement according to the invention (Fig. 4a) is the fact that measurements can be carried out "continuously" in this way with acoustic measuring devices (Figure 4a (0)-(3)) since the surface of the vibrating object will assume the equilibrium starting position again after the measurement has been carried out and will therefore again be available for carrying out a measurement. In this "continuous" method of measurement, a membrane which is permeable to the ligand to be measured but impermeable to the "leaving agent" is preferably installed between the vibrating object and the point at which the sample to be examined is added (Fig. 4b) , Such a membrane should have pores having a diameter of <50 nm and may in principle be any marketed membrane like a cellulose acetate membrane. In this way, the abovementioned medium which is advantageously circulated only needs to contain the respective ligand in a constant concentration since the "leaving agent" cannot escape from the space between the membrane and the surface of the vibrating object.

A particular variant of the first-mentioned achievement is to use a vibrating object whose surface situated at the liquid/solid interface is provided with a multiplicity of particulate carriers which contain specific binding partners for the ligand, said specific binding partners being provided, in addition, with a "leaving agent" in the form of a particle reversibly bound to said specific binding partners (see Figure 5) •

Further it is pointed at the possibility that after the introduction of the sample to be investigated in the above mentioned embodiments according to the invention latex particles having a diameter of 50-1000 nm and provided with antibodies for the ligand in question are applied under the formation of a sandwich-complex with the ligand, which is attached to the specific binding partner which in its turn is bound directly or indirectly to the surface of the vibrating object.

In relation to the achievements cited above for eliminating the known disadvantages in the prior art relating to acoustic methods of measurement it is stressed that they all relate to increasing the change in mass per unit surface of the vibrating object by using latex spherules having a diameter of 50-1000 nm, so that there is "unity of invention¹'.

The vibrating object used in the method according to the invention is advantageously a quartz crystal whose two mutually opposite surfaces are coated with a metal layer, preferably gold or silver. The thickness of such a metal layer is normally 10-100 nm and can be applied in a known manner such as vapour deposition and the like.

Within the scope of the invention, many types of carrier particles can be used as particulate carriers. Advantageously, the carriers used are latex spherules having a diameter in the range of, for example, 10-1000 nm, preferably 50-200 nm. Examples of suitable materials are, inter alia, (monodisperse) polystyrene latex, polymethyl methacrylate latex, a silica latex or a latex of a conducting polymer such as polypyrrole and polyacetylene. Specific examples of latices are Unisphere latex particles having a diameter of 50 nm (type 10) or 100 nm (type 11) (supplied by Brunschwig Chemie B.V., The Netherlands) and "Polybead" polystyrene microspheres having a diameter of 50, 100 or 200 nm (Polysciences Corp. Niles/Illinois, USA).

The "degree of coverage" of the sensor surface of the quartz crystal which is used as vibrating object and which is provided with the loaded particulate carriers (see Figure 2) can vary considerably. For example, the degree of coverage depends on the size of the carrier particles or on the type of specific binding partner, such as antibodies and DNA strands. In general, it can be stated that the degree of coverage is 50-99- ->% • For the sake of completeness it is pointed out that "degree of coverage" of the sensor surface with the spherules is understood to mean the ratio of the sum of the surface of the cross section of the "spherical" carriers or of the spherical particles reversibly bound to the specific binding partners multiplied by a factor of 100 and divided by the total surface of the sensor surface.

"Degree of loading" of the spherules with the specific binding partners is understood to mean the ratio of the loaded surface, i.e. the surface covered with the specific binding partners, of the spherules multiplied by a factor of 100 and divided by the total surface of the spherules in the monolayer or submonolayer. In general, the degree of loading is 10-90JU.

Many types of compounds can be used as ligands to be determined in a sample or the specific binding partners complimentary thereto. The antigen has already been mentioned above as ligand and the antibody specific for the antigen as specific binding partners. For an incomplete summary of usable ligands or specific binding partners, reference is made to Table A below.

The immobilisation of the specific binding partners on the latex spherules is a technique known per se. For example, a general description of the technique is given in "Microparticle Immunoassay Techniques" (Seradyn Inc., ed. Galloway and Hicks, Particle Techno¬ logy Division, P.O. Box 1210, Indianapolis, IN 46206, USA).

The carriers loaded with specific binding partners can then be bound to the metal layer of the quartz crystal used as vibrating object by means of adsorption or by means of applying a thin layer of glue or a layer of another type of adhesive by a dipping technique, spin coating or other generally known deposition techniques.

For the sake of completeness, attention is drawn to the fact that the above procedure can, if desired, be reversed by first carrying out the adhesion of the spherules to the metal layer of the quartz crystal used as vibrating object and then the immobilisation of the specific binding partners on the spherules acting as carriers. The particulate carriers can also be applied by means of a flat coating, such as a polystyrene coating, to the metal surface, i.e. the particulate carriers are indirectly immobilised on the metal surface. ^•

The invention furthermore relates to an object to be set vibrating and suitable for use in an acoustic measuring device such as a "bulk acoustic wave" or a "plate wave" measuring device, comprising an object to be set vibrating, preferably a quartz crystal, two mutually opposite surfaces of which are coated with a metal layer of, advantageously, gold or silver, one of the metal layers or an unmetallised part of the object to be set vibrating being provided with a quantity of latex spherules having a diameter of 50-1000 nm as particulate carriers which have been applied thereto and which contain specific binding partners for the ligand

(Figure 2) , or a quantity of specific binding partners which have been applied thereto and which are provided with a "leaving agent" in the form of a particle reversibly bound to said specific binding partners being a ligand on a latex spherule having a diameter of 50-1000 nm (Figure 3). or a quantity of particulate carriers which have been applied thereto and which contain specific binding partners, the specific binding partners also being provided with a "leaving agent" in the form of a particle reversibly bound to said specific binding partners being a ligand on a latex spherule having a diameter of 50-1000 nm (Figure 5) •

CAPTIONS Figure 1: Diagrammatic representation of the method known from the prior art (Thompson et al., loc.cit.).

Figure 2: Diagrammatic representation of the method according to the invention in which the specific binding partner is located on particulate carriers attached to the surface of the vibrating object.

Figure 3= Diagrammatic representation of the method according to the invention, the "leaving agent" being located on the specific binding partners of the ligand attached to the surface of the vibrating object and being removed by the ligand by means of a displacement reaction.

Figure 4a: Diagrammatic representation of the method according to the invention, an established equilibrium between the specific binding partners attached to the surface of the vibrating object (1) and the ligand and "leaving agent" contained in the medium being shown. Figure 4a (0-3) These figures show instants in time in relation to a measurement, where Figure 4a(0) : represents the equilibrium phase at the instant t₀;

Figure 4a(l): represents the sampling at the instant i;

Figure 4a(2) : represents the equilibrium phase modified by the sampling at the instant t₂; and

Figure 4a(3): represents the restored equilibrium phase (as at t₀) at the instant t₃.

Figure 4b: Diagrammatic representation of the method according to the invention, in which a membrane

(2) which is permeable to the ligand but impermeable to the "leaving agent" is installed between the surface of the vibrating object (1) with specific binding partners attached thereto and the point at which the sample to be examined is added. Figure : Diagrammatic representation of the method according to the invention, in which the specific binding partner for the ligand is located on the spherules attached to the surface of the vibrating object and in which the specific binding partner of the ligand itself is also provided with a "leaving agent", which "leaving agent" is remov by the ligand by means of a displacement reaction

Figure 6: Cross section of the sensor container containing quartz crystal as object to be set vibrating, which:

(1) represents the quartz crystal;

(2) represents sealing material; and

(3) represents an electrical contact.

Figure 7-' Frequency diagram obtained with a BAW sens comprising a quartz crystal provided with go electrodes on which there is HSA (adhesive laye and PS spherules. The numbers 1-2 in Figure 7 ha the following meaning:

(1) addition of anti-HSA (concentration 690 nmol/1);

(2) addition of Tween-20 (0.1# v/v) , as a resul of which desorption occurs.

Figure 8: Frequency diagram obtained with a BAW sensor. Thi is based on a clean quartz crystal provided wit gold electrodes. The numbers 1-2 in Figure 8 hav the following meaning:

1) addition of anti-HSA (625 nmol/1);

2) removal of the solution and rinsing with PB (0.01 mol/1).

Figure 9 '. Frequency diagram obtained with a BAW sensor. Thi was based on a clean crystal provided with gol electrodes. The numbers 1-4 in Figure 9 have t following meaning:

1) addition of HSA (concentration: 1.5 μmol/1 (as the result of the failure of th stirrer, a rise in frequency is observed a a consequence of the occurrence of a interface effect) ;

2) stirrer on again, as a result of whic adsorption occurs;

3) removal of the solution and rinsing with PB (0.01 mol/1); 4) addition of a suspension of PS spherules

(diameter: 100 nm; concentration 0.0025 solids).

Figure 10: Frequency diagram obtained with a BAW sensor. This was based on the quartz crystal after the treatment described in Figure 9- The numbers 1-9 in Figure 10 have the following meaning:

1) increase in the concentration of a suspen¬ sion of PS spherules (diameter: 100 nm) to 0.025 solids;

2) removal of the suspension and rinsing with PBS (0.01 mol/1);

3) addition of HSA (concentration: 1.5 μmol/1);

4) removal of the solution and rinsing with PBS (0.01 mol/1);

5) addition of anti-TNP (concentration: 150 nmol/1) ;

6) addition of anti-HSA (concentration: 150 nmol/1) ; 7) addition of HulgG (concentration:

150 nmol/1) ;

8) increase in the concentration of anti-HSA to 300 nmol/1;

9) increase in the concentration of anti-HSA to 1700 nmol/1.

The invention is explained in greater detail with reference to the examples below.

In carrying out the BAW sensor measurements, oscillators were used which were contained in a metal housing. The oscillator was fed with a 15 volt direct current supply and consumed approximately 25 milliampere. The supply was connected to the oscillator with the aid of a miniature twin connector. The oscillator signal was fed from a 50 ohm buffer control circuit via a binary connector (BNC) to an external counting device. The oscillator had only one adjustment device which adjusted both the amplitude and the phase. It was found that, with good, clean crystals and good connections, the oscillator normally functioned without adjustment, even if the quartz crystal sensor was located in liquid on one side.

The quartz crystal sensors were installed in the base of a stainless-steel housing, which housing was itself connected to the top of an oscillator with the aid of a two-pin connector. In this way, a very compact measuring device was created which ensured a low noise because of the effective electrical screening and also the absence of long cables to connect the sensor to the oscillator. A cross section of the sensor housing is shown in Figure 6. During all the measurements, the frequencies of one or two oscillators, A and B, were periodically measured at known instants and stored on a hard disc of a PC. For this purpose, the oscillators were connected to a two-channel counting device (HP 5334B) which was regulated and read out with the PC. These data were used to produce frequency diagrams such as, for example, Figures 7-10.

The oscillator(s) and the stirrer(s) were kept in a sealed climatic chamber during the test. The rest of the device, for example the oscilloscopes, counting device, supply and PC were outside the climatic chamber.

Example I

A) Application of base laver loaded with latex spherules to the quartz crystal, the latex spherules being provided with HSA (human serum albumin)

A coating of HSA (human serum albumin; Sigma A 3782; concentration 1 mg/ml) which acted as an adhesive layer was applied to a quartz crystal (area: 0.5 cm²) provided with a gold electrode (area: 0.05 cm², applied by means of a vapour deposition technique). After an incubation time of half an hour, the coated gold surface was rinsed with PBS (phosphate buffered saline solution) . Polybead polystyrene (2.5 solids) carboxylated microspheres (Polysciences Corp. Niles. 111., USA) (200 nm; PS spherules) were then applied to the coated gold layer. After 10 min, the metal surface was rinsed with PBS.

In a subsequent step, 2 ml of a 2% solution of carbodiimide prepared by dissolving 45 mg of carbodiimide in 2 ml of PBS was applied dropwise to the metal layer obtained, which was provided with spherules, after which an incubation was carried out for 6 hours at room temperature (20°C) and at a relative humidity of 100%.

The product obtained was then rinsed with a borate buffer (Polysciences Corp., Niles/Ill., USA). In the subsequent step, protein (HSA, Sigma A 3782) was applied (1 mg/ml; the surface of the gold layer coated with spherules was completely covered) , after which storage was carried out for one night at 100% relative atmospheric humidity. After this period, the product was again rinsed with PBS.

Finally, the unreacted groups originating from carbodiimide were blocked with the aid of an excess of 0.1 M ethanolamine solution for 30 min at room temperature.

B) Measurement of the frequency shift with anti-HSA (α-HSA) : see Figure 7.

A solution of α-HSA (Sigma A 1151) (690 nmol/1) was applied to the coated gold layer of the quartz crystal (see point (1) of Figure 7)-

From Figure 7 it can be deduced that a Δf of 1600 Hz occurs for α-HSA (690 nmol/1) on a BAW crystal coated with 0.2 μm PS spherules.

COMPARISON EXAMPLE: see Figure 8

A) To apply the HSA to a quartz crystal provided with a gold layer, the procedure is as described in Example I, but with the difference that the step of applying the PS spherules is not carried out. B) For the purpose of measuring the frequency shift, an α-

HSA solution (625 nmol/1; Sigma A 1151) (see point (1) of Figure 8) was applied to the gold layer of the crystal surface coated solely with HSA. From Figure 8 it can be deduced that a frequency shift of only 600 Hz occurs. Compared with the two frequency shifts above (1600 Hz for a gold layer coated with PS spherules and 600 Hz for a gold layer not coated with PS spherules) it can be stated that the sensitivity has increased by a factor of approximately 3-

Example II

This example illustrates the immunological specificity of anti-HSA with respect to HSA applied to the surface of the quartz crystal. This surface was first coated with PS spherules as particulate carriers to increase the surface density of HSA. In the text below, reference is made to the associated frequency diagrams obtained with the underlying BAW sensor (Figures 9 and 10) . The solution above the BAW sensor was continuously stirred.

A solution of HSA (Sigma A 3782; 1.5 μmol/1) was applied to the surface of a quartz crystal (area: 0.5 cm²) provided with gold electrodes (area: 0.05 cm²) (point (1) of Figure 9)- The solution was removed and the crystal was rinsed with 0.01 mol/1 PBS (point (2) of Figure 9)- A suspension of PS spherules (diameter 100 nm; Polysciences Corp., Niles/Ill., USA; 0.0025% solids) was then applied with the object of coupling the PS spherules, the applied HSA coating acting as adhesive layer (point (3) of Figure 9)-

The concentration of the PS spherule suspension was then increased to 0.025% solids (point (1) of Figure 10). The suspension was then removed and the crystal was rinsed with 0.01 mol/1 PBS (point (2) of Figure 10). The crystal surface coated with PS spherules was completely covered with HSA by adding a solution of HSA (Sigma A 3782; 1.5 μmol/1) (point (3) of Figure 10). After rinsing with 0.01 mol/1 PBS (point (4) of Figure 10), a solution of anti-TNP (150 nmol/1) was added, and this did not result in a frequency shift (point (5) of Figure 10) in contrast to the addition of anti-HSA (Sigma A 1151; 150 nmol/1) (point (6) of Figure 10), which indicates specific adsorption. Control addition of HulgG (Nordic Immunological Laboratories b.v.; 150 nmol/1) resulted in a very low nonspecific adsorption (point (7) of Figure 10). Further increases in the concentration of anti-HSA to, respectively, 300 nmol/1 (point (8) of Figure 10) and 1700 nmol/1 (point (9) of Figure 10) brought about a further frequency shift. From Figure 10 it can be deduced that specific adsorption occurs of anti-HSA at the crystal surface coated with HSA and very low nonspecific adsorption of the other antibodies, anti-TNP and HulgG.

Claims

CLAIMS 1. Method of determining a ligand in a liquid sample by bringing the sample into contact with a vibrating object which is accommodated in an acoustic measuring device and whose surface is provided with specific binding partners for the ligand to be determined, and analysing the frequency shift or the change in the acoustic propagation velocity or the change in the amplitude or the phase change measured at the vibrating object and due to the formation of a complex between the ligand and the specific binding partner, characterised in that, in said method, a vibrating object is used whose surface situated at the liquid/solid interface is provided with a multiplicity of latex spherules having a diameter of 50-1000 nm as particulate carriers which contain specific binding partners for the ligand.

2. Method of determining a ligand in a liquid sample by bringing the sample into contact with a vibrating object which is accommodated in an acoustic measuring device and whose surface is provided with specific binding partners for the ligand to be determined, and analysing the frequency shift or the change in the acoustic propagation velocity or the change in the amplitude or the phase change measured at the vibrating object and due to the formation of a complex between the ligand and the specific binding partner, characterised in that, in said method, a vibrating object is used whose surface situated at the liquid/solid interface contains a multiplicity of specific binding partners which are provided with a "leaving agent" in the form of a particle reversibly bound to said specific binding partners and being a ligand on a latex spherule having a diameter of 50-1000 nm.

3- Method of determining a ligand in a liquid sample by bringing the sample into contact with a vibrating object which is accommodated in an acoustic measuring device and whose surface is provided with specific binding partners for the ligand to be determined, and analysing the frequency shift or the change in the acoustic propagation velocity or the change in the amplitude or the phase change measured at the vibrating object and due to the formation of a complex between the ligand and the specific binding partner, characterised in that, in said method, a vibrating object is used whose surface situated at the liquid/solid interface contains a multiplicity of specific binding partners for the ligand which are partly provided with the ligand and the "leaving agent" in the form of a ligand on a latex spherule having a diameter of 50-1000 nm which ligand is reversibly bound to said specific binding partners in accordance with an established equilibrium with a medium (competition reaction) which is advantageously circulated and which contains a constant concentration of the ligand and the "leaving agent", said established equilibrium being temporarily shifted, after introducing a sample to be examined, resulting in a corresponding frequency shift.

4. Method according to Claim 3. characterised in that the specific binding partners for the ligand are bound to a multiplicity of latex spherules having a diameter of 50-1000 nm as particulate carrier, attached to the surface of the vibrating object.

5- Method according to one or more of Claims 1-4, characterised in that after introducing the sample to be examined latex spherules having a diameter of 50-1000 nm provided with antibodies for the ligand are applied under the formation of a sandwich-complex with the ligand attached to the specific binding partner, which specific binding partner is bound directly or indirectly to the surface of the vibrating object.

6. Method according to Claim 3 or 4, characterised in that a membrane which is permeable to the ligand to be measured but impermeable to the "leaving agent" is installed between the vibrating object and the point at which the sample to be examined is added.

7- Method according to Claim 1, characterised in that the specific binding partners bound to the particulate carrier are also provided with a "leaving agent" in the form of a particle reversibly bound to said specific binding partners being a ligand on a latex spherule having a diameter of 50-1000 nm.

8. Method according to one or more of Claims 1-7, characterised in that a quartz crystal whose two mutually opposite surfaces are coated with a metal layer is used as vibrating object.

9- Method according to Claim 8, characterised in that a quartz crystal whose two mutually opposite surfaces are coated with a gold or silver layer is used as vibrating object.

10. Method according to Claim 1, 4 or 7, characterised in that the particulate carriers are applied to the metal surface by means of a flat coating, such as a polystyrene coating.

11. Method according to one or more of Claims 1-5 or 7. characterised in that latex spherules are used which have a diameter in the range of 50-200 nm.

12. Method according to one or more of Claims 1-5 and 7. characterised in that a polystyrene latex or a polymethyl methacrylate latex is used.

13- Method according to one or more of Claims 1-5 and 7. characterised in that a silica latex is used.

14. Method according to one or more of Claims 1-5, 7 an 11-13, characterised in that the "degree of coverage" of the surface of the object with the particulate carriers or particles reversibly bound to the specific binding partners is 50-99-5%-

15- Method according to one or more of Claims 1-5, 7 and 11-13, characterised in that the "degree of loading" of the surface of the particulate carriers with the specific binding partners is 10-90%.

16. Object suitable for use in an acoustic measuring device whose two mutually opposite surfaces are coated with a metal layer of, advantageously, gold or silver, one of said metal layers being provided with a quantity of latex spherules having a diameter of 50- 1000 nm as particulate carriers which have been applied thereto and which contain specific binding partners for the ligand. 17- Object suitable for use in an acoustic measuring device whose two mutually opposite surfaces are coated with a metal layer of, advantageously, gold or silver, one of said metal layers being provided with a quantity of specific binding partners for the ligand which are provided with a "leaving agent" in the form of a particle reversibly bound to said specific binding partners and being a ligand on a latex spherule having a diameter of 50-1000 nm. 18. Object according to Claim 16, in which the specific binding partners bound to the particulate carrier are also provided with a "leaving agent" in the form of a particle reversibly bound to said specific binding partners and being a ligand on a latex spherule having a diameter of 50-1000 nm.

19- Object according to one or more of Claims 16-18, characterised in that the object is a quartz crystal.