WO2007034508A1

WO2007034508A1 - An ultra-sensitive method for detection and quantification of substance

Info

Publication number: WO2007034508A1
Application number: PCT/IN2006/000369
Authority: WO
Inventors: Ajay Kumar Sood; Ajay Singh Negi
Original assignee: Indian Institute Of Science
Priority date: 2005-09-19
Filing date: 2006-09-18
Publication date: 2007-03-29

Abstract

The present invention relates to an ultra-sensitive method for detection and/or quantification of an analyte in a sample, comprising steps of: contacting the analyte present in the sample with an analyte-counterpart; and applying an electric field in a direction perpendicular to the plane of the electrode for a period sufficient to cause the analyte-counterpart to bind to analyte for detection and quantification of the analyte; an apparatus for detection and/or quantification of an analyte in a sample comprising: electrodes; means for applying electric energy to the sample in perpendicular direction; and means for detecting read-out signal; and lastly a kit for detection and/or quantification of an analyte in a sample comprising: electrodes; means for applying electric energy to the sample in perpendicular direction; counterpart of analyte capable of recognizing the analyte means for detecting read-out signal; and manual of instructions.

Description

AN ULTRA-SENSITIVE METHOD FOR DETECTION AND QUANTIFICATION OF SUBSTANCE

FIELD OF THE INVENTION The present invention relates to an ultra sensitive assay. More particularly the present invention is in relation to an ultra-sensitive method for detection and quantification of a substance and also provides an apparatus and a kit for detection of desired analyte in a sample.

BACKGROUND OF THE INVENTION

There is a large number of latex particle based immunoassay tests used for diagnostic purposes. The presence of antigen is often determined by the agglutination of the antibody coated particles. The tests are reliable up to a finite concentration of the antigen. Below a certain concentration of antigen, the agglutination does not occur.

PRIOR ART OF THE INVENTION

Agglutination is a diffusion governed process and hence quite slow. In order to enhance the rate of agglutination and improve the sensitivity of tests, especially at low concentrations in pico and femto molar range, many techniques have been tried, some of which are set out here below:

(a) The non-cavitating standing wave ultrasound has been used to increase the sensitivity of different latex agglutination tests because of the increased rate of particle collision as antibody coated particles are forced into the pressure nodal regions. (Grundy et al, J of Immunological Methods, vol 165, p 47, (1993) and

Ellis et al, J Med. Microbiol., vol 49, p 853, (2000)).

(b) Microfluidic systems with controlled flow of small volumes of fluids is another approach to enhance the interaction between antigen and antibodies. (Verpoorte, Electrophoresis, vol. 23, p 677, (2002) and Kricka, Clinical Chemistry, vol 44, p 2008, (1998). sensitivity- sensitivity will depend upon the detection method and so we are not quoting it here.)

(c) Coplanar electric field has also been used to form chains of colloidal particles thereby enhancing the rate of latex agglutination reactions (Song et al, Analytical Chemistry, vol. 66, p. 778, (1994). Sensitivity will depend upon the detection method and so we are not quoting it here.)

All the aforesaid methods of the prior art have their own inherent limitations. For example, in coplanar fields, the particles form chains. In a chain the number of neighbours is at the maximum two. In other words, it is only possible to form arrays of the particles (like a pearl chain). Hence, in case even if there are more number of binding sites on the antibody coated particle, one can not improve the sensitivity any further. Microfluidic systems need a lot of precise engineering which is not always available at hand. Thus, there is a need in the art to provide a method and system whereby the sensitivity of the assay is increased and the material in a sample may be detected even at low concentrations.

OBJECTS OF THE PRESENT INVENTION: The principal object of the present invention is to develop an ultra-sensitive method for detection and/or quantification of an analyte in a sample.

Another object of the present invention is to develop an apparatus for detection and/or quantification of an analyte in a sample.

Yet another object of the present invention is to develop a kit for detection and/or quantification of an analyte in a sample.

STATEMENT OF THE INVENTION:

The present invention relates to an ultra-sensitive method for detection and/or quantification of an analyte in a sample, comprising steps of (a) contacting the analyte present in the sample with an analyte-counterpart; and (b) applying an electric field in a direction perpendicular to the plane of the electrode for a period sufficient to cause the analyte-counterpart to bind to analyte for detection and quantification of the analyte; an apparatus for detection and/or quantification of an analyte in a sample comprising (a) electrodes; (b) means for applying electric energy to the sample in perpendicular direction; and (c) means for detecting read-out signal; and lastly a kit for detection and/or quantification of an analyte in a sample comprising (a) electrodes; (b) means for applying electric energy to the sample in perpendicular direction; (c) counterpart of analyte capable of recognizing the analyte (d) means for detecting readout signal; and (e) manual of instructions. DESCRIPTION OF THE INVENTION

The present invention relates to an ultrasensitive method for detection and/or quantification of an analyte in a sample, comprising steps of: a) contacting the analyte present in the sample with an analyte-counterpart; b) applying an electric field in a direction perpendicular to the plane of the electrode for a period sufficient to cause the analyte-counterpart to bind to analyte for detection and quantification of the analyte.

In yet another embodiment of the present invention the method is capable of detecting amounts of analyte ranging upto pico Molar concentrations.

In still another embodiment of the present invention the analyte and analyte-counterpart agglutinate to form large clusters in the region of electric field .

In still another embodiment of the present invention the analyte is a biological macromolecule.

In still another embodiment of the present invention the analyte is an antigen.

In still another embodiment of the present invention the antigen is selected from a group comprising microbe, enzyme, toxin, hapten, and other foreign particles.

In still another embodiment of the present invention the analyte-counterpart is selected from a group comprising protein and carbohydrate.

In still another embodiment of the present invention the analyte-counterpart is an antibody.

In still another embodiment of the present invention the method can be used to detect analyte in diseased conditions.

In still another embodiment of the present invention the method is used for detection and/or quantification of Rheumatoid Factor in a sample comprising steps of: a) contacting the Rheumatoid factor present in the sample with an RF Reagent; b) applying an electric field in a direction perpendicular to the plane of the electrode for a period sufficient to cause the RF Reagent to bind to Rheumatoid Factor for detection and quantification of the Rheumatoid Factor.

The present invention relates to an apparatus for detection and/or quantification of an analyte in a sample comprising:

- electrodes,

- means for applying electric energy to the sample in perpendicular direction; and

- means for detecting read-out signal.

In still another embodiment of the present invention the electrodes of the aforementioned apparatus are two glass plates coated with conducting indium-tin- oxide.

In still another embodiment of the present invention the means for electric energy in the aforementioned apparatus is a function generator combined with a potentiometer used to regulate the strength of applied sinusoidal voltage which is read on a multimeter.

The present invention relates to a kit for detection and/or quantification of an analyte in a sample comprising:

- electrodes,

- means for applying electric energy to the sample in perpendicular direction;

- counterpart of analyte capable of recognizing the analyte,

- means for detecting read-out signal, and - manual of instructions.

Brief description of the accompanying drawing

Figure 1 shows analytes trapped between substrate to which the analyte-counterpart are attached causing them to agglutinate.

Figure 2 shows an experimental set up for implementing the invention.

Figure 3 shows clusters for three concentrations of RNase both with and without field after 30 minutes of mixing the streptavidin coated particles and biotinylated RNase. Figure 4 shows a plot with variation of mean area per cluster versus concentration of

RNase added.

Figure 5 shows the agglutination in Rhelax RF reagent in the presence of positive control, both in the presence and absence of electric field.

Figure 6 shows the variation of standard deviation of pixel intensity with concentration of the positive control.

Figure 7a shows illustration of variant of the method using ELISA.

Figure 7b shows illustration of variant of the method, wherein two electrodes are dipped into the vial to enable effective formation of analyte-counterpart and analyte complex and development of read-out signal.

Contrary to the approaches of the prior art, the invention provides a technique whereby presence of even minimal amounts of analyte may be detected in a sample. The invention is based on the finding that when electric field is applied in Z-direction (perpendicular to the confining plates) the analyte and analyte-counterpart agglutinate and tend to form large clusters in the region of the electric field, leading to detection of even minimal amounts of desired analyte in a given sample.

The applicant has found that under the influence of a perpendicular electric field, the substrates to which the analyte-counterpart are attached, are caused to aggregate, and as a result the analyte is trapped between the substrate. A diagrammatic representation of the mechanism of action is shown in Figure 1. Since in a cluster, the number of neighbours of a particle are more, this method facilitates the formation of analyte and analyte-counterpart links better and hence gives excellent and very high sensitivity.

The term 'analyte' as used herein may include any substance which is desired to be detected in a given sample. It may be an antigen, antibody, sugar, hapten, toxin or any other such material. The sample may be such as urine, saliva, blood, synovial fluid, cerebrospinal fluid and any other biological material, whether natural or synthetic. The term 'analyte-counterpart' is used to denote any material that is capable of recognising the analyte. For instance, if the analyte is an antigen, the analyte-counterpart may be an antibody or an appropriate carbohydrate. The 'binding partner' or substrate is any particle or material to which the analyte-counterpart is attached, either by covalent or non-covalent linkages. It includes latex, polystyrene, gold, silver, platinum or any other polymeric or fibroses particles onto which the analyte-counterpart may be attached.

The results may be read or detected by any detectable signal and observed employing techniques such as chemiluminescence, colour, radioactivity, reflectance, fluorescence, birefringence, changes in optical density (at specific or broad wavelength), measurement of absorbance of the transmitted light (Turbidimetry), measurement of scattered light at a small angle to the forward direction (called Nephelometry), scanning laser microscopy, visual observation and digital imaging.

The invention may be implemented through a number of different embodiments, but the underlying principle is to apply an electric field in the Z-direction and detect even minimal amounts of the analyte in a sample. Some of these embodiments are illustrated below:

A) An analyte-counterpart is prepared to the analyte in question. For example, if the analyte is an antigen, then antibodies to that antigen are prepared and kept ready.

Thereafter, polymeric materials such as latex bodies are coated with the said analyte- counterpart prepared, followed by preparation of an admixture comprising the coated material and analyte in the sample. The admixture so prepared is a colloidal dispersion, which is placed on a solid surface (the surface being pre-coated with a conductive material) and subject to an electric field (in Z-direction) such that analyte-counterparts binds to the analyte and the results are detected by an appropriate read-out signal.

Optionally, the unbound bodies and reaction solution may be washed off after a certain period so that only immobilized complexes are left for observation of read-out signal.

B) Yet another variant may be an ELISA (enzyme-linked-assay) wherein first analyte- counterpart is attached to a first substrate. A second analyte-counterpart and enzyme attached to a second substrate is also provided. The first analyte-counterpart is introduced into a sample and after some time the second analyte-counterpart is introduced followed by an electric field applied perpendicular to the plane of the sample for a period sufficient to cause the first-analyte-counterpart and second-analyte- counterpart (with enzyme) to agglutinate and produce a detectable readout signal. The method is illustrated in Figure 7(A).

C) In a variant method, the patient's sample may be collected in vials and analyte- counterpart attached to the substrate is added to the vial. Then the two electrodes are dipped into the vial to enable effective formation of analyte-counterpart and analyte complex and development of read-out signal. This embodiment is illustrated in Figure 7(B).

In all the aforesaid aspects, the electric field applied may be alternating current or may be battery operated.

In another aspect, the invention provides an apparatus useful for detecting the presence of an analyte in a sample, comprising:

- a conducting electrode; source of electric energy, including a means for applying electric energy to the electrode in a direction perpendicular to the electrodes.

In yet another aspect, the invention provides a kit for detection of the presence of an analyte in a sample. The kit would vary depending on the kind of test employed. However, in general, the kit may comprise:

- conducting surfaces to act as electrodes,

- a source and means for applying electric energy to the sample in perpendicular direction;

- counterpart of analyte capable of recognizing analyte and substrate for binding of analyte-counterpart,

- means for detecting read-out signal, and

- manual of instructions.

Thus, the principle of the invention may be implemented in various forms and by various variant embodiments, all of which are deemed and intended to fall within the scope of the invention. The invention is further elaborated with the help of following examples. However, these examples should not be construed to limit the scope of the invention. Example 1:

An embodiment setting out an example experimental set up for implementing the invention as shown in Figure 2. Antibody coated microspheres and the analyte mixture was sandwiched between two glass plates (17) coated with conducting indium-tin oxide (ITO) (18). The two plates are separated by insulating spacers of 75 μm thickness and the cell was sealed to prevent evaporation and flow of the suspension. A function generator combined with a potentiometer (25) may be used to regulate the strength of applied sinusoidal voltage which may be read on a multimeter (16). A polarising microscope (30) (in transmission mode) combined with CCD video camera (40) may be used to image the particles. The observations are recorded on a video recorder (45). The setup also has a computer with monochrome image grabber card PCI-1411 (National Instruments, USA) (50) which can digitize images at a rate of 25 frames per second. The images captured with this card may be analyzed to quantify the extent of agglutination.

Example 2:

Using the experimental set up as shown in figure 2, the following experiments were conducted:

(a) Model System: The colloidal suspensions of polystyrene particles (diameter = 0.95 μm) coated with streptavidin with desired volume fractions (0.2 %) were prepared by adding deionized water to the stock suspension (1 %) bought from M/s Bangslab, USA. As a model antigen, biotinylated ribonuclease A (RNAse) in water was used. Each molecule of RNAse had on average five biotin molecules. This biotinylated RNAse acts as the linkage between two streptavidin coated latex particles.

(b) Commercial Test: This was done on a commercial latex agglutination test kit for rheumatoid factor (Rhelax RF test, Tulip Co., Goa, India). This kit comes with a Rhelax RF reagent which is a suspension of polystyrene latex particles coated with suitably modified Fc fraction of IgG. The reagent is standardised to detect » 10 IU/ml of RF or more. The kit also contains a positive control which we have used as the antigen. Rhelax RF reagent was diluted to 1/10 of initial concentration to facilitate viewing under microscope.

One volume of antibody coated particles was mixed with one volume of different dilutions of the analyte containing the antigen and this mixture was sandwiched between the plates. The experiments were done both with and without field. A voltage of 2 V was chosen in case of streptavidin coated particles and a voltage of 3 V was used in case of Rhelax test. Example 3:

Results and analysis:

A) Biotinylated RNAse test:

In the absence ■ of RNAse (antigen), the streptavidin coated particles do not show appreciable aggregation without field and in the presence of applied voltage of 2 volts. However, in the presence of antigen, upon application of a small electric field of frequency 1 IcHz the streptavidin coated colloidal particles start to aggregate into randomly shaped two-dimensional clusters on the bottom plate. The size of clusters formed varied with the concentration of the biotinylated RNAse added. Fig. 3, shows clusters for three concentrations of RNAse both with and without field after 30 minutes of mixing the streptavidin coated particles and biotinylated RNAse. As shown in figure 3, the concentration of biotinylated RNAse increases from top to bottom in the figure. It can be seen that bigger aggregates form as the concentration of biotinylated RNAse increased. Clusters at different concentrations of biotinylated RNAse as shown. Fig. 3 (a) and (b) shows no RNAse, Fig.3 (c) and (d) has 1 pM and Fig. 3 (e) and (f) has 1 nM of RNAse. Also, it is seen that for the same concentration of biotinylated RNAse, the aggregates are much bigger when an electric field is applied thereby indicating that electric field helps in agglutination.

At each concentration of RNAse, five images of different clusters have been taken and using image analysis, calculated out the average area per cluster. The variation of average area per cluster with the concentration of RNAse added is plotted in the Fig. 4. The mean area cluster has been plotted against the concentration of RNAse and biotin (in picomolar). The mean area has been calculated by taking average over 5 images. It is evident from the plot that while the mean area does not increase much in the absence of electric field, the presence of electric field produces a tremendous improvement in the agglutination of latex particles.

It is clearly evident from the graph that at concentration of 10 pM, the average area per cluster increases rapidly in case when the field is on. As shown in the said figure, at IpM itself, when field is applied, certain material is observed. At lOpM the mean cluster area observed is greater; when concentration of RNAse and biotin is further increased in the sample to 10OpM, the mean cluster area is 1000 and the same goes on rising as the sample concentration rises. This is illustrative of the fact that even at IpM concentration of sample, the analyte may be detected in the sample, which is very high as compared to currently available methods.

B) Rhelax RF test: In case of Rhelax RF test, the aggregates formed were not confined to the bottom plate as shown in Fig. 5. When there was no clustering, the image was uniformly bright but with clustering, the image started showing inhomogeneous brightness. The standard deviation was calculated in the pixel intensities of the image and that was plotted as the measure of aggregation in the system as shown in Fig. 6. The mean of standard deviations of pixel intensities of 30 images at each concentration was taken with and without field. The images were taken after 10 minutes of mixing the RF reagent and the positive control with and without field. It can be seen that in the electric field facilitates the aggregation even at a much higher dilution of the positive control. The sensitivity has been enhanced by a factor of atleast 500.

Figure 5 shows the agglutination in Rhelax RF reagent in the presence of positive control, both in the presence and absence of electric field. Agglutination is more prominent at higher concentration of the positive control. Presence of electric field aids in the agglutination. Figure 6 shows the variation of standard deviation of pixel intensity with concentration of the positive control. When there is no clustering, the image is uniformly bright but with clustering, the image starts showing inhomogeneous brightness. Thus the standard deviation in the pixel intensities of the image is a direct measure of aggregation in the system. The plot indicates that aggregation increases with the concentration of the positive control and presence of electric field improves the aggregation for the same concentration of positive control. ADVANTAGES OF THE INVENTION

(a) It is very easy to setup, simple to use and versatile as it can be applied to any readout method. It can be employed for agglutination tests for many diseases.

(b) It is highly sensitive.

(c) The sensitivity of this test can be improved by using other methods for read outs such as fluorescence, turbidimetry, etc.

(d) By the method of the invention, the sensitivity may be improved at least by a factor of 500.

Claims

WE CLAIM:

1. An ultrasensitive method for detection and/or quantification of an analyte in a sample, comprising steps of: a) contacting the analyte present in the sample with an analyte-counterpart; b) applying an electric field in a direction perpendicular to the plane of the electrode for a period sufficient to cause the analyte-counterpart to bind to analyte for detection and quantification of the analyte.

2. The method as claimed in claim 1, wherein the method is capable of detecting amounts of analyte ranging upto pico Molar concentrations.

3. The method as claimed in claim 1, wherein the analyte and analyte-counterpart agglutinate to form large clusters in the region of electric field .

A. The method as claimed in claim 1, wherein the analyte is a biological macromolecule.

5. The method as claimed in claim 1, wherein the analyte is an antigen.

6. The method as claimed in claim 1, wherein the antigen is selected from a group comprising microbe, enzyme, toxin, hapten, and other foreign particles.

7. The method as claimed in claim 1, wherein the analyte-counterpart is selected from a group comprising protein and carbohydrate.

8. The method as claimed in claim 1, wherein the analyte-counterpart is an antibody.

9. The method as claimed in claim 1, wherein the method can be used to detect analyte in diseased conditions.

10. The method as claimed in claim 1, wherein the method for detection and/or quantification of Rheumatoid Factor in a sample comprising steps of: a) contacting the Rheumatoid factor present in the sample with an RF

Reagent; b) applying an electric field in a direction perpendicular to the plane of the electrode for a period sufficient to cause the RF Reagent to bind to Rheumatoid Factor for detection and quantification of the Rheumatoid Factor.

11. An apparatus for detecting and/or quantification of an analyte in a sample comprising: o electrodes, o means for applying electric energy to the sample in perpendicular direction; and o means for detecting read-out signal.

12. The apparatus as claimed in claim 11, wherein the electrodes are two glass plates coated with conducting indium-tin-oxide.

13. The apparatus as claimed in claim 11, wherein the means for electric energy is a function generator combined with a potentiometer used to regulate the strength of applied sinusoidal voltage which is read on a multimeter.

14. A kit for detection and/or quantification of an analyte in a sample comprising: o electrodes, o means for applying electric energy to the sample in perpendicular direction; o counterpart of analyte capable of recognizing the analyte, o means for detecting read-out signal, and o manual of instructions.