Description

State of the art

Alzheimer's disease (AD) is a progressive brain disorder characterized by memory loss and confusion [ 1 , 2]. The incidence of AD doubles every 5 years in patients over 60 years of age and currently affects more than 35 million patients worldwide [3, 4]. The development of the disease can start even 20 years before the first signs of cognitive functions of the brain [5]. The brain in Alzheimer's disease is characterized by high levels of certain proteins from S I 00 protein family, including the S 100B protein [6]. It is a calcium-binding protein secreted by astrocytes. The increased concentration of this protein was found in serum and cerebrospinal fluid of patients with AD [7, 8]. It is believed that the observed higher levels of S 100B protein concentrations are important in the pathogenesis of neurodegenerative processes [6, 9]. S I OOB protein exerts both neurotrophic and potentially neurotoxic effects within the body, depending on its concentration [10]. This means that it affects very clearly the conversion of dispersed amyloid non-fibrilar deposits into axonal neuritic forms and, in consequence, to the development of the disease itself [6]. Moreover, the level of S 100B protein may be useful in order to distinguish the severity of, or monitor the development of dementia in the course of the disease [2]. Thus, S 100B protein can be considered as a potential biomarker for AD [2, 6, 9, 1 1 -13].

Most, if not in all, of the cellular effects associated with S 100B protein (neurotrophic and neurotoxic effects) are mediated by Receptor for Advanced Glycation Endproducts (RAGE) [14, 15]. RAGE is a multiligand receptor consisting of three extracellular domains V, C I , C2, a transmembrane helix and a short (42 amino acids) tail [8, 16].

RAGE plays an important role in certain human pathologies, such as diabetes, stroke, cancer, chronic inflammation and neurodegenerative disorders, including Alzheimer's disease [17, 18, In many publications RAGE is described as a common extracellular receptor of S 100B protein [ 14, 20-21]. The interaction between RAGE and S 100B protein is strictly dependent on the presence of Ca ²⁺ [22]. Binding of Ca ²⁺ opens the structure of S 100B and visualization of the protein-protein interaction sites. Thus, only S 100B protein bound to Ca ²⁺ with exposed binding site is capable of forming a complex with the RAGE [22].

Conventional techniques for the determination of potential biomarkers in AD comprise surface plasmon resonance (SPR) [19, 23], ELISA [6], and liquid chromatography [24]. However, these methods have several disadvantages: they are time-consuming, expensive, require specialized equipment and highly skilled workers. Electrochemical sensors are analytical devices which are promising alternative to the currently used detection systems. Ease of use and relatively low cost make them gain an importance in the study of proteins and nucleic acids and their interactions [24].

The invention solves an extremely important problem for diagnosis of a stroke, cancer, chronic inflammation or a neurodegenerative disorder, especially Alzheimer's disease. Until now, different diagnostic methods of the disease have been used, but none of these methods provide reliable and accurate diagnosis. The present invention provides means for performing a minimally invasive diagnostic method of providing reliable data to allow the diagnosis.

Summary of the Invention

The invention relates to a biosensor comprising a gold electrode covered with a redox active layer comprising a DTPA-Cu(II) complex and fixed domains selected from the RAGE domains in combination with the His tag and the mutant His ₆ -RAGE VC 1 domain. Preferably, the His-tagged RAGE domain is His ₆ -RAGE C2 domain. Biosensor is applicable to the determination of level of S 100B protein, fragment or analogue thereof in a sample. Preferably, the biosensor is for use in the diagnosis of stroke, cancer, chronic inflammation or a neurodegenerative disorder by measuring the S 100B protein with the use of the biosensor, especially for use in the diagnosis of Alzheimer's disease.

The present invention also relates to a method for producing the biosensor, wherein said method comprises modifying a clean gold electrode with thiol derivative of pentetic acid (diethylene triamine pentaacetic acid - DTPA) and N-acetylcysteamine (NAC) and then allowing to form a complex with Cu ²⁺ ions, wherein said method comprises immobilizing the His ₆ -RAGE domain, His ₆ -RAGE C2 domain and mutant His ₆ -RAGE VC 1 domain, following by conditioning of the electrode.

Detailed Description of the Invention

The biosensor of the present invention comprises redox-active layer comprising a complex of DTPA-Cu (II). This complex plays two functions: a link between the surface of the gold electrode and immobilized His -RAGE domains and transducer in the intermolecular recognition process. Presented biosensor was used to observe the interaction between RAGE receptor domain VC 1 and S 100B protein. To observe the effects of the above, the electrochemical method is used - a square wave voltammetry. The interaction between the protein immobilized on the surface of the electrode and a specific compound in an aqueous solution causes a change in electrochemical properties of DTPA derivatives in the complex of Cu(II) present on the surface of the gold electrode. In result, a change in the availability of ions present in the solution to the the redox - active centers occurs. This results in the generation of the analytical signal. In order to verify whether the observed analytical signal is the result of intermolecular recognition process between domain His ₆ -RAGE VC 1 and S 100B protein, His ₆ -RAGE C2 domains and mutant His ₆ -RAGE VC 1 domain were used, which do not have the ability to bind S 100B protein. The selectivity of the present biosensor was tested in the presence of a constant concentration of the other ligand, as well as in the presence of diluted human plasma.

The invention is illustrated in the drawing, in which

Fig. 1. Representative voltammetric curves recorded for the electrode modified by the layer of NAC/DTPA-Cu(II) - His ₆ -RAGE VC1 (a) in the presence of a buffer and in the presence of a concentration of the S 100B protein (b) 1 pM; (c) 5 pM; (d) 10 pM; (e) 15 pM; (f) 20 pM. A- measurements performed in a buffer containing Ca ⁺ ; B- measurements performed in buffer without the presence of Ca ²⁺ . Measurement conditions: 0.1 M KC1, scan speed of 100 mV/s. Fig. 2. The charts show the following dependence (I _n -I ₀ )/I ₀ [%] on the concentration of the C [pM] of S 100B protein in buffer composed of 50 mM TRIS, pH 7.4; (A) with the addition of Ca ²⁺ (■) without the addition of Ca ²⁺ . Examples

Example 1

Cleaning of the gold electrodes

Immediately before modification a gold electrode was cleaned mechanically by polishing in aqueous suspensions of aluminum oxide (alumina) having particles of a diameter of 0.3 and 0.05 μιη, for 5 minutes each. In the next step, the electrode was cleaned electrochemically in 0.5 M solution of potassium hydroxide by the potential sweep in the range of -1200 mV to -400 mV (vs. Ag/AgCl as a reference electrode) at a rate of 100 mV/s, the number of cycles: 3, 50 and 5. Then, the electrode was placed into 0.5 M solution of sulfuric acid (VI) with a sweep potential ranging from -300 mV to 1500 mV at 100 mV/sec, number of cycles: 3, 10, and 5. Finally, the electrode surface was refreshed in KOH solution using 10 cycles.

Modification of gold electrodes

Cleaned gold electrodes were washed with ethanol-water (volume ratio 4: 1) and then dipped in a solution of diethylene triamine pentaacetic acid (DTP A) and N-acetylcysteamine (NAC) (in the molar ratio 1 : 100) in the above mixture at 1 8 h at 4°C. Then, the electrode was rinsed with ethanol-water, water, chloroform and chloroform-methanol, and immersed in a 1.0 x 10 ^"3 M solution of copper acetate (II) in a mixture of chloroform-methanol (volume ratio 1 : 1) for 3 h at room temperature. In this step, there is a process of forming a complex of Cu ions with diethylene triamine pentaacetic acid (DTPA) attached to the surface of the gold electrode to form a molecular connector. Following this modification step, the electrode was rinsed in the following order: a mixture of chloroform-methanol, methanol, water and 0.1 M potassium chloride solution and then was stored in 0.1 M potassium chloride solution until use. Modifying solutions were placed each time in glass tubes with a diameter of 8 mm with a rounded bottom. After placing the electrodes in modifying solutions the tubes were secured with Teflon tape and Parafilm in order to prevent evaporation of the solvent.

Immobilization of His ₆ -RAGE VCl

10 μΐ of a His -RAGE VCl solution in the buffer at a concentration of 10 mM was spotted for 24 h at 4°C with cover on the pre-rinsed with buffer surface of modified electrode. After the modification the electrode was rinsed, and then conditioned in an appropriate buffer: 300 mM Tris, 500 mM NaCl, pH 7.4 in the case of electrodes with an immobilized VC l domain of RAGE protein. Conditioning time was 16 hours.

Example 2

The study of the interaction between the His^-RAGE VCl domain immobilized on the surface of the electrodes and the SWOB protein present in an aqueous solution

10 μΐ of a solution containing the protein S 100B in buffer composed of 50 mM TRIS, 25 mM CaCl ₂ , pH = 7.4 or in the same buffer composition but without CaCl ₂ was spotted on the gold electrode surface modified with layer of NAC/DTPA-Cu(II) - His ₆ -RAGE VCl reversed upside down. Electrode was protected against water evaporation by covering with Eppendorf tube. The incubation time was 30 min.

Then the electrode was washed with a buffer composed of 50 mM TRIS, 25 mM CaCl ₂ , pH 7.4, or buffer having the same composition, but without 25 mM CaCl ₂ .

After rinsing the electrode with an appropriate buffer, the electrode was rinsed with a solution of 0.1 M C1 and placed in the electrochemical cell. Electrochemical measurements were carried out in the presence of 0.1 M KG. Formation of a complex His ₆ -RAGE VCl- S100B on the electrode surface results in a reduction of oxidation and reduction current of the redox- active center of Cu(II) and the shift of potential position of the maximum current in the direction of negative potential. These changes were proportional to the concentration of S100B protein. For measuring the technique of square- wave voltammetry was used. Representative results obtained for the interaction between the His ₆ -RAGE VC l domain immobilized on the electrode and S100B protein contained in the solution were shown in Fig. 1.

Characteristic reduction in redox Cu (II)/Cu (I) current and offset of the potential towards negative values with increasing concentration of analyte is the evidence for the interaction between the immobilized His ₆ -RAGE VC l domain and the S IOOB protein in the presence of Ca ²⁺ (Fig. 1 A , Fig. 2). Absence of Ca ²⁺ caused a significant reduction of the above effects (Fig. I B, Fig. 2). References

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