WO1997004313A1

WO1997004313A1 - Analytical electroimmunoassays and electroassays for biological recognition

Info

Publication number: WO1997004313A1
Application number: PCT/ES1996/000151
Authority: WO
Inventors: Agustin Costa Garcia; Maria Begoña GONZALEZ GARCIA; Cesar Fernandez Sanchez
Original assignee: Universidad De Oviedo
Priority date: 1995-07-18
Filing date: 1996-07-18
Publication date: 1997-02-06
Also published as: ES2102970B1; AU6419596A; ES2102970A1; ES2149104A1; ES2149104B1

Abstract

Analytical electroimmunoassays and electroassays for biological recognition, which combine the proper selectivity of immunological reactions with the sensitivity of electrodico-absorptive properties of an electrode of carbon paste which is used as support and transducer for the analytical signal. Application to biomedical analysis.

Description

Analytical electroimmunoassays and biological recognition electroassays

The invention, which has application in the field of biomedical analyzes, relates to analytical electroimmunoassays and biological recognition electroassays that combine the selectivity of immunological reactions with the sensitivity of the electrode-adsorptive properties of a carbon paste electrode that It is used as support and transducer of the analytical signal. It also refers to a method to increase the sensitivity of determinations for low levels of concentration of the protein material that covers the electrode.

Enzyme-modified carbon pulp electrodes have recently been used as electrochemical biosensor transducers (Domínguez Sánchez, P., O'Suliivan, CLK., Miranda Ordieres, AJ, Tuñón Blanco, P., Smyth, MR, 1 994, Chim. Actu. 291: 349-356).

This easily crafted electrode material (graphite powder + paraffin) developed and experienced by Adams et al. (Adams, R. N.,

"Electrochemistry at solid electrodes", 1 969, 280-283) at the beginning of the 1960s and widely used since then, has adsorption characteristics on different organic substances that make it possible to carry out coating studies of different materials reliably on its surface. These coatings result in low concentrations of the aforementioned organic substances can be easily quantified, if in their structure there were electroactive groups or electrochemical marks capable of being oxidized or reduced by applying a certain potential to the electrode on which they are located (Cortina Villar , JV, Costa García, A., Tuñón Blanco, P., 1 993, Tatania

40: 325-331), (Cortina Villar, J.V., Costa García, A., Tuñón Blanco, P., 1 992,

Anal. Chim. Minutes 256,231).

A large number of ELISA methods that use electrochemical detection as the basis for quantification are found in the recent literature analytical (Thompson, RQ et al., 993, Anal. Chim. Acta 271: 223-229); (Yu, Z. et al., 1 994, Journal of Pharm. And Biomc. Anal. 1 2: 787-793); (Masson, M. et al., 1 995, Anal. Chem. 67: 1 245-1 267); (Gal la Salle et al., 1 995, Anal. Chem. 67: 1 245-1 253) made with vitrified carbon electrodes showing the advantages and disadvantages of these newly developed immunoelectrotechnologies.

Thus, it has been shown that concentrations of the order of 1 0 ²¹ M of alkaline phosphatase-labeled IgG can be detected amperometrically with a carbon electrode in an ELISA with amperometric detection (Thompson, RQ et al., 1 993, Anal. Chim. Minute 271: 223-229) and other ELISAs with similar characteristics have been developed with low quantification limits for different analytes.

The vast majority of electro-enzyme-immunoassays have been constructed on vitrified carbon electrodes and have the great disadvantage of reproducibility, mainly due to the great difficulty involved in the renewal of the electrode surface of a vitrified carbon electrode.

To date, no immunoelectroenzymatic assay that has been verified on carbon paste electrodes is known.

Recent studies by the inventors have shown that biotinylated albumin, streptavidin and immunoglobulin coatings can be carried out on this type of electrodes with a rigorous control of the coating, if they are conveniently marked with electrochemical or enzymatic markings. These results have made it possible to have developed a multisensor method that, based on the fundamentals of ELISA techniques in general and on the adsorptive characteristics of properly treated carbon paste, presents application possibilities as general as those with the same ELISA methods, but with much higher analytical characteristics. The Success lies in a strict control of the electrode surface, which means that a surface with very similar electrodesic-adsorptive properties is used in each experiment.

According to the invention, a device and method could be designed both to quantify biotin and to quantify avidin, and since biotin is one of the most universal haptens and macromolecule markers, through it any carbon paste could be attached molecule or structure capable of being labeled with biotin.

Therefore, objects of the invention are analytical electroimmunoassays and biological recognition electroassays, which use a carbon paste electrode as a transducer of the analytical signal, and which have a protein part adsorbed on the surface of the electrode for biochemical interactions. .

Said analytical electroimmunoassays and biological recognition electroassays use the same electrode surface in each test, which is recoverable after a convenient chemical-electrochemical treatment, with small gold particles and gold colloidal particles being provided as a necessary mark for monitoring the biological reaction , and the use of silver as a sensitizing element of the analytical signal, as well as alkaline phosphatase as an enzymatic brand for monitoring biological interaction.

Another object of the invention is a method of cleaning and activating the electrode surface comprising:

- washing in an aqueous solution, - imposing a positive potential for a suitable time, and a method of fixing the protein part on the electrode surface, once cleaned and activated, comprising:

- washing in a suitable pH solution,

- controlled adsorption on the electrode surface of the protein part. In said method, the use of gold particles as electrochemical marks is provided by:

- controlled albumin adsorption to eliminate nonspecific adsorption,

- biological interaction between the protein part absorbed on the electrode and the analyte that competes with the same analyte adsorbed on gold particles or marked with them,

- development of the analytical signal as a result of the oxidation of the gold particles fixed on the electrode by subjecting it to a positive potential in a medium of chlorides and then reducing them in the same medium by performing a voltammetric scanning towards negative potentials.

Preferably, the method uses silver to sensitize the analytical signal by:

- cleaning and activation of the electrode surface,

- controlled coating of the protein part on said surface, - removal of nonspecific adsorption with albumin,

- fixation of gold particles through the biological reaction on the electrode,

- deposition, catalyzed by gold particles, of metallic silver on said particles when the electrode is introduced into a silver solution containing a suitable reducer,

- oxidation of metallic silver deposited in an iodide medium by subjecting the electrode to a potential sweep.

Also preferably, the method uses alkaline phosphatase by:

- elimination of nonspecific adsorption by introducing the electrode into an albumin solution,

- biological interaction between the protein part absorbed on the electrode and the analyte that competes with the same analyte labeled with alkaline phosphatase,

- washing for the elimination of enzymes not fixed by the biological reaction,

- introduction of the electrode into the enzyme substrate suitable for the development of the voltammetric signal. The analytical electroimmunoassays and biological recognition electroassays according to the invention could use other electrodes or ultramicroelectrodes as transducers of the analytical signal, as well as using other complexing or precipitating means, such as iodide, which are used as a sensitizing medium in the oxidation of the silver.

The analytical electroimmunoassays and biological recognition electroassays of the invention would be usable for the quantification of antigens, haptens and antibodies.

A first exemplary embodiment of the invention comprises a carbon pulp electrode that acts as a transducer of the analytical signal and a protein part adsorbed on the pulp that acts as the site of biochemical interactions.

The basic difference in relation to a typical biosensor is that after each experience it will be necessary to clean the electrode surface and renew the protein layer.

The transducer consists of a carbon paste electrode, whose basic scheme can be seen in the examples illustrated in the attached Figures 1 and 2.

The carbon paste is prepared, for example, by mixing in a mortar 5 grams of graphite powder and 1.8 ml of paraffin, which is placed in a lower drain of a Teflon cylinder 1 which contains a rod of one conductive metal 2 which acts as an electrical contact between the carbon paste 3 and the measuring instrument (for example, a potentiostat) not shown.

Once the carbon paste 3 has been introduced into the lower drain of the Teflon cylinder 1, the outer surface thereof is polished on a sheet of paper and then subjected to the following electrode pretreatment: The electrode is introduced into a solution of NH ₃ 1.0 M, this solution is stirred and a potential of + 1 .00 V is imposed on the electrode for a period of 1 20 seconds.

This electrode pretreatment prior to any determination or test is absolutely essential. It is the one that always works with an electrode surface with similar characteristics and that reproducibilities higher than 96% are achieved.

The coating of the protein part is described with reference to Figure 1.

Once the electrode surface of the carbon paste 3 is pretreated, it is washed in a phosphate buffer solution of pH 8.0 and then the electrode is introduced into another phosphate buffer solution of pH 8.0 which is at the same time 10 ^"4 M in Streptavidin is maintained for 5 minutes in this streptavidin solution, which is stirring slightly, thus fixing a layer of streptavidin 5 molecules on the carbon paste surface 3.

Then, to avoid risks of nonspecific adsorption, the electrode, once its surface has been modified with streptavidin, is introduced 5 minutes in another buffer solution of pH 8.0 with 1% albumin. In this way, albumin molecules 4 are also fixed on the electrode surface and block the centers of nonspecific adsorption.

The electrode thus modified becomes a biosensor that can be applied both to the quantification of biotin and to the quantification of avidin from an unknown sample.

In the case of biotin quantifications, the electrode thus prepared would be introduced into the unknown biotin sample of pH 8.0 during a 60 minute incubation time in which the biotin molecules would bind to a part of the adsorbed streptavidin molecules about him electrode (always in excess). The electrode is removed from this first incubation and introduced into another pH 8.0 buffer solution containing biotinylated albumin adsorbed on colloidal gold particles or marked with small gold particles, and held in this solution for another 60 minutes in order to that the streptavidin molecules that had remained unreacted with the biotin molecules in the sample now react with the adsorbed biotin molecules on the colloidal gold particles.

After this second incubation period, the electrode is removed and taken to the quantization cell containing a 0.1 M HCI solution in addition to a platinum auxiliary electrode and a saturated Ag / AgCI reference electrode. In this medium the electrode processes that will be described later for colloidal gold are caused, so that the recorded signal will depend directly on the number of biotiny colloidal gold particles 6 attached to the electrode surface through the streptavidin-biotin interactions. colloidal gold Therefore, the analytical signal will be inversely related to the concentration of biotin in the sample.

With this methodology it is possible to quantify biotin samples with concentrations between 1 ^0'9 M and 1 0 ^"6 M.

If avidin were to be quantified with this same design, the streptavidin adsorbed on the electrode should be competed with the avidin of the sample in solution, for the same concentration of biotinylated albumin adsorbed on colloidal gold also in solution. As there was less concentration of avidin in the sample, more biotiny colloidal gold particles could bind to the streptavidin modified electrode surface. Therefore, also in this case the analytical signal would be inversely related to the concentration of anaite in the sample, in this case avidin.

Referring to the scheme in Figure 2, the methodology would be similar to that described for the scheme in Figure 1, but now the coating protein that would be adsorbed on the previously activated carbon paste 3 would be biotinylated albumin 4 'and albumin 4. The concentration used of biotinylated albumin would be 1 0 ^"4 M and, as in the scheme of Figure 1, could also be used to quantify both biotin and avidin, however, in this case streptavidin adsorbed onto 6 'colloidal gold would be used to obtain the analytical signal.

The quantification limits and the dynamic linear ranges resemble those found for the scheme in Figure 1.

The electrochemical brand is colloidal gold. This brand is used for the first time as the basis of the voltammetric signal for monitoring this interaction. Biotin or avidin control will be carried out indirectly through the oxidation-reduction process of colloidal gold, which consists of the following:

Colloidal gold adsorbed through the interaction is perfectly adhered to the carbon paste surface, since a mechanical stirring of the 2000 rpm solution is unable to release any colloidal gold particles from the electrode.

The development of the analytical signal is based on the following electrode processes:

1 ^or Au _coloid .- _(ads) + 4CI ^"- 3E> Auci _{4 (ads)}

2nd .- AuCI _{4 (ads)} + 3e ^" > Au + 4CI ^'

The first process is caused by subjecting the electrode to a potential of + 1 .25 V in relation to an Ag / AgCI reference electrode saturated with KCI, in a 0.1 M HCI medium.

The second process is the one that gives rise to the analytical signal. Once the first process has been verified, the same electrode is subjected to a sweep of potentials from + 1 .25 V towards less positive potentials in which a voltammetric peak around + 0.43 V appears as a result of reduction of adsorbed AuCI ₄ .

In accordance with the invention and as another objective, a sensitization of the methodology followed has also been developed, in order to reach lower levels of biotin or avidin concentration, which is based on the known fact and applied in microscopy (Dandcher , G., Worgaard, JOR, Histochem J., 1 983, Cytochem, 31: 1 394-1 398) that gold particles may increase in size by depositing metallic silver on them. This can also be done within the method of the invention, with the advantage that quantifying Ag on a solid electrode is much easier than quantifying gold.

It consists of oxidizing the deposited silver in the presence of a complexing or precipitating agent of the silver.

The methodology consists of the following phases:

one . Cleaning and activation of the electrode surface. This implies the regeneration of the electrode when positive potentials are applied in a neutral environment.

2. Controlled coating of the protein part on the electrode surface, which consists of adsorbing on said surface the protein necessary for biological interaction.

3. Elimination of nonspecific adsorption, which is achieved by introducing the electrode into an albumin solution.

4. Gold particle fixation, which takes place through the biological reaction between the protein part fixed in the electrode and the analyte fixed or marked with the gold particles.

5. Deposition of metallic silver catalyzed by gold particles. It is achieved by introducing the electrode that already has adsorbed on its surface the gold particles in a silver solution and a reducer, usually hydroquinine, which causes the gold particles to be surrounded by metallic silver.

6. Achievement of the analytical signal. This is achieved by oxidizing the metallic silver deposited on the gold particles by introducing the electrode into a solution of potassium iodide and make a voltammetric scan towards positive potentials.

In the presence of I, an oxidation process is obtained at a potential of -0.23 V against an electrochemically reversible Ag / AgCI reference electrode.

Ag ° + I - le ^' > Agí E = -0.23

It can also be ensured that on this carbon paste transducer, sandwich-type enzyme electro-immunosensor designs work with the same reproducibility benefits as biotin / avidin biosensors.

Starting from a conventional design of enzymatic electroimmunoassay, a design that is not new as a whole, and using carbon paste as an immunosensor transducer, the reproducibility that is obtained is above 96% making this design have analytical utility in terms of Your applications

Alkaline phosphatase-labeled immunoglobulin coatings have also been achieved as an enzyme and reproducibility is achieved when the electrode is pretreated as follows:

If the electrode is used for the first time, its surface is subjected to a potential of + 1 .6 V for 1 0 minutes in a pH 9 phosphate buffer. This step is done only once. And then as a pretreatment that is repeated for each test, the following is done: 1 ^o ) Wash for 2 minutes in 0.1 M NaOH with stirring. 2 ^o ) Imposition of a potential of + 1.5 V for 5 minutes in a pH 9 phosphate buffer.

This electrode pretreatment makes the same surface of carbon paste can be used in successive tests with similar electrode-adsorptive properties, and these tests comprise the following stages: A) Controlled coating of the protein part on the electrode surface, which consists of adsorbing on said surface the protein necessary for biological interaction.

B) Elimination of nonspecific adsorption that is achieved by introducing the electrode into an albumin solution.

C) Fixation of alkaline phosphatase as a result of the biological reaction between the coating of the protein part and the analyte labeled with said enzyme.

D) Introduction of the electrode in an aqueous solution of neutral pH in order to eliminate enzymes not fixed by biological interaction.

E) Introduction of the electrode in which the enzymatic label has been immobilized on substrates such as naphthylphosphate, which develop a suitable voltammetric signal by effecting on the electrode a sweep of potentials towards positive potentials.

The basic instrumentation for practicing the present invention would consist, for example, of an electrolytic cell for three electrodes (working, auxiliary and reference) and a potentiostat plus a wave generator or a polargraph.

An electroimmunotechnology according to the invention would compete with known immunotechnologies, such as RIA, EMIT, ELISA, etc., of which the most representative and most frequently used today would be ELISA, a technique against which electroimmunotechnology of the invention has, among others, the following advantages:

- Faster than the ELISA technique: from the preparation of the electrode to the obtaining of the analytical signal the elapsed time would be approximately 2 hours and 1 5 minutes. An ELISA would require from the preparation of the incubation plates until obtaining the analytical signal times always exceeding 24 hours.

- More economical: the electrode material is a relatively very material cheap, and the same electrode can be used for more than 20 analyzes approximately.

- It can be as sensitive as ELISA methods, with intrinsic possibilities that once developed will allow greater sensitivity than the aforementioned ELISAs.

- Presents greater linear dynamic ranges; in the case of biotin, the concentration range may range from 10 ^'9 M and 1 0 ^"6 M.

- The percentage of data dispersion is always less than 4%, while the percentages of ELISA methods are always above 8%, mainly due to the fact that each test is done in a different well and the number of washes needed.

- It is an easily automated technology that can be miniaturized with the use of ultra-microelectrodes for the use of microvolumes.

- The instrumentation necessary for the development of the analytical signal is a relatively cheap instrumentation (potentiostat).

Claims

1.- Analytical electroimmunoassays and biological recognition electroassays, which use a carbon paste electrode as a transducer of the analytical signal.

2.- Analytical electroimmunoassays and biological recognition electroassays, which have a protein part necessary for biochemical interactions adsorbed on the surface of the electrode.

3.- Analytical electroimmunoassays and biological recognition electroassays, according to the previous claims, which use the same electrode surface in each test, which is recoverable after a convenient chemical-electrochemical treatment.

4.- Analytical electroimmunoassays and biological recognition electroassays, according to the previous claims, which use small gold particles and colloidal gold particles as a necessary mark for monitoring the biological reaction.

5.- Analytical electroimmunoassays and biological recognition electroassays, according to the previous claims, which use silver as a sensitizing element of the analytical signal.

6.- Analytical electroimmunoassays and recognition electroassays, according to the previous claims, which use alkaline phosphatase as an enzymatic mark for monitoring the biological interaction.

1.- A method of cleaning and activating the electrode surface that includes:

- washing in an aqueous solution, - imposition of a positive potential for an appropriate time.

8.- A method of fixing the protein part on the electrode surface once cleaned and activated, according to the previous claim, which comprises:

- washed in a solution of suitable pH,

- controlled adsorption on the electrode surface of the protein part.

9.- A method, according to the previous claim, that uses gold particles as electrochemical marks, according to claim 4, and comprising:

- controlled adsorption of albumin to eliminate non-specific adsorptions,

- biological interaction between the protein part absorbed on the electrode and the analyte that competes with the same analyte adsorbed on gold particles or labeled with them,

- development of the analytical signal as a consequence of the oxidation of the gold particles fixed on the electrode, by subjecting it to a positive potential in a chloride medium, and subsequently reducing them in the same medium by performing a voltammetric scan towards negative potentials.

1 0.- A method that uses silver to sensitize the analytical signal, according to claim 5, and comprising:

- cleaning and activation of the electrode surface,

- controlled coating of the protein part on said surface,

- elimination of non-specific adsorptions with albumin, - fixation of gold particles through the biological reaction on the electrode,

- deposition, catalyzed by the gold particles, of metallic silver on said particles by introducing the electrode into a silver solution containing a suitable reducer, - oxidation of the metallic silver deposited in an iodide medium by subjecting the electrode to a sweep of potentials.

1 1.- A method, according to claim 8, that uses alkaline phosphatase, according to claim 6, and comprising:

- elimination of non-specific adsorptions by introducing the electrode into an albumin solution, - biological interaction between the protein part absorbed on the electrode and the analyte that competes with the same analyte labeled with alkaline phosphatase,

- washing to eliminate enzymes not fixed by the biological reaction,

- introduction of the electrode into the appropriate enzymatic substrate for the development of the voltammetric signal.

1 2.- Analytical electroimmunoassays and biological recognition electroassays, according to claims 2 to 1 1, which use other electrodes or ultramicroeielectrodes as transducers of the analytical signal.

1 3.- Analytical electroimmunoassays and biological recognition electroassays, according to claim 1 0, using other complexing or precipitating means, such as iodide, which are used as a sensitizing medium in the oxidation of silver.

14.- Use of analytical electroimmunoassays and biological recognition electroassays, according to claims 4 to 6, for the quantification of antigens, haptens and antibodies.