Aptamer, aptasensor comprising aptamer, and use of the aptamer for selective detection of manganese ions

Abstract

The object of the present invention is an aptamer capable of specific detection of manganese ions which has a nucleotides sequence described in SEQ. ID NO. 1. An aptasensor with affinity to manganese ions Mn2+ which comprises a gold working electrode (1) coated with a receptor layer (2) comprising aptamers (3) and filler (4) molecules and an anionic indicator (5) that is present in the solution. A method of obtaining aptasensor comprising a step of forming the receptor layer (2) by incubation of the gold electrodes (1) in an aptamer (3) solution for 2 h and step b) of incubation with a filler (4) solution for 1 h to eliminate non- specific interactions on the surface of the electrode. The use of the aptamers as a receptor layer of a biosensor for selective in situ detection of manganese ions by electrochemical techniques.

Description

[0001] Aptamer, aptasensor comprising aptamer, and use of the aptamer for selective detection of manganese ions

[0002] The present invention relates to an aptamer, an aptasensor containing the aptamer, and the use of the aptamer for detection of manganese ions.

[0003] The correct functioning of organisms depends on the availability of drinking water, a source of which is groundwater and surface water, including rivers, lakes as well as natural and artificial water reservoirs. The drinking water is subjected to contamination due to the constant development of areas e.g., urbanization, construction, agriculture and mining. Among the most dangerous pollutants are heavy metals, including manganese. Manganese is used in the production of among others batteries, fertilizers or ceramics. Although manganese is an essential element for the correct growth and development of the central nervous system in the human body, its elevated concentration may lead to various types of diseases, including cell death in focal cerebral ischemia, epilepsy or neurodegenerative diseases such as Huntington's or Alzheimer's disease. Among the commonly used analytical techniques for detecting manganese levels are energy dispersive X-ray fluorescence spectrometry (EDXRF), atomic absorption spectrometry (AES), and atomic absorption spectrometry coupled to microwave plasma (ICP-AES). Due to the necessity to use sophisticated apparatus, the presence of an experience operator and the time-consuming nature of the analysis, it is necessary to develop new solutions for detecting manganese ions.

[0004] The solution can be the use of biosensors comprising bioreceptor layers with high selectivity towards manganese ions. The specificity of the receptor layer is determined by properties of biological molecules and in case of detecting manganese ions, there are reports of the possibility of using a genetically modified protein specific for lanthanides, that is lanmodulin. There is also a report in literature (M. Mizunuma et al., 2023) disclosing the use of SELEX technique and the identification of an aptamer sequence with a high content of guanine bases, which can constitute the receptor layer of the fluorescent biosensor (aptasensor) for the detection of manganese ions.

[0005] Aptamers are short, single-stranded sequences of RNA or DNA oligonucleotides that change their spatial conformation as a result of an interaction with analytes of various classes. The aptamers are used, among others, as drug carriers and their capability of functional groups introduction allows them to be attached to a specific surface or groups constituting optical or electrochemical tags which means that they can also be used as receptor layers of biosensors. The aptamers are identified by using the SELEX process, which involves generating a pool of sequences of up to 1015of the different sequences using combinatorial chemistry methods, which sequences are then incubated with a specific analyte. Sequences not attached to the analyte are removed, whereas in the further step the detachment of the analyte from the aptamer molecule occurs. The set of the sequences is then amplified by PCR method and is subjected to another round of interactions with the analyte, ultimately leading to a narrowed group of aptamer sequences showing the greatest affinity to the selected analyte. A limited group of sequences is then subjected to sequencing as well as their structural stability is determined. It should be emphasized, however, that the length of the aptamer sequence plays a significant role in the degree of interaction with the selected analyte. The MnG4Cl sequence obtained by M. Mizunuma's team (July 2023) has 40 nucleotides, which may favour the occurrence of the non-specific interactions with components of the real samples, including other metal ions. Although selectivity studies were carried out, they concerned analysis using the circular dichroism technique for high concentrations of interfering ions - 100 pM and did not consider, among others the analysis for lead ions. It should also be noted that over the course of more than 30 years, thanks to the use of the SELEX technique, it was possible to identify aptamer sequences for a limited number of metal ions, including lead, potassium, copper and cadmium ions.

[0006] The biggest challenge is to achieve high selectivity of the receptor layers made by aptamer sequences already at the step of using the SELEX technique. In turn, the usefulness of the aptamer receptors layers can be confirmed by coupling them with an appropriate detection technique characterised by high sensitivity, low detection limits, as well as low cost and simplicity of the realisation of the measurement. These features are ensured by the use of electrochemical techniques such as voltammetry and impedance spectroscopy.

[0007] The aim of the invention is to solve the problem of the lack, in the state of the art, of an optimal aptamer sequence that interacts specifically with manganese ions and which at the same time demonstrates selectivity towards selected interfering ions at a level below the maximum permitted concentration of manganese ions in water samples in accordance with the WHO guidelines (1.82 pM) as well as the Polish guidelines (0.91 pM). The aptamer sequence according to the invention, which can be used as a receptor layer, shows increased selectivity by adjusting the sequence length as well as the composition of the nucleotides constituting the sequence. Gist of the Invention

[0008] The object of the invention is an aptamer capable of a specific detection of manganese ions characterised by the fact that has a nucleotides sequence as shown in SEQ. ID NO.l.

[0009] Moreover, the aptamer is characterised by the fact that is modified with a disulphide group.

[0010] Preferably, the aptamer according to claim 1 is characterised by the fact that the disulfide group modification is on the 5’ terminal.

[0011] Further object of the invention is an aptasensor having an affinity to manganese ions Mn2+, characterised by the fact that comprises a gold working electrode 1 coated with a receptor layer 2 that comprises aptamers 3 according to the invention, and filler 4 molecules as well as an anionic indicator 5 that is present in the solution. Preferably, the aptasensor is an electrochemical sensor.

[0012] Preferably, the aptasensor receptor layer 2 is covalently bonded is covalently bonded to a surface of the electrode. Preferably, the filler 4 in the aptasensor is 6-mercapto-l -hexanol.

[0013] Preferably, the anionic indicator 5 for generating a current signal is anthraquinone-2-sulfonic acid sodium salt.

[0014] Preferably, the aptasensor is characterised by the fact that the affinity of the receptor layer 2 to manganese ions is 64 nM.

[0015] Even more preferably, the aptasensoris characterised by the fact that the range ofits dynamic linear response is from 50 nM to 1 pM.

[0016] Another object of the invention is method of obtaining an aptasensor comprising steps of: a) formation of a receptor layer 2 by incubation of the gold electrodes 1 in an aptamer 3 solution for 2 h, b) incubating in a filler 4 solution for 1 h to eliminate unspecific interactions on a surface of the electrode.

[0017] Preferably the method is characterised by the fact that the concentration of the solution in step a) is 4 pM. Preferably, the method is characterised by the fact that the filler 4 is 6- mercapto-1 -hexanol. Preferably, the method is characterised by the fact that the concentration of the filler 4 is 4 pM.

[0018] Another object of the invention is a use of the aptamers as a receptor layer of a biosensor for in situ selective detection of manganese ions by utilizing electrochemical techniques. Preferably, the use is characterised by the fact that manganese ions are detected in liquid samples.

[0019] More preferably, the use is characterised by the fact that manganese ions are detected in a water.

[0020] Preferably, the use is characterised by the fact that manganese ions are detected in a culture medium.

[0021] Explanation of the Figures of the Drawing

[0022] The object of the invention is described in the examples of the realisation in the drawing in which:

[0023] Fig i shows a schematic structure of the receptor layer (2) comprised of DNA aptamers (3) and 6-mercapto-l -hexanol filler (4).

[0024] Fig.2 shows a schematic operation of the receptor layer (2) comprised of DNA aptamers (3) and 6-mercapto-l -hexanol filler (4) after incubation of the electrodes in the solution comprising manganese ions and the redox indicator (5).

[0025] Fig.3 shows a dynamic response of the miniaturized electrodes, from Metrohm Dropsens company, modified with Al sequence (working electrode no. 1) and with reference sequence polyT (working electrode no. 2). 6-mercapto-l -hexanol was used as a blocking agent to the working electrodes.

[0026] Fig.4 shows a comparison of a current responses of the miniaturized electrodes, from Metrohm Dropsens company, modified with Al sequence (working electrode no. 1) and with reference sequence polyT (working electrode no. 2) as well as with blocking agent being 6- mercapto-1 -hexanol, after incubation with manganese ions and a mixture of interfering ions.

[0027] Fig.5 shows a comparison of a current response for the gold disk electrodes modified with Al sequence (4 pM) and blocking agent being 6-mercapto-l -hexanol (4 pM), which were incubated with 100 nM manganese ions (in 50 mM MES, pH 6.0) in the presence of the redox indicators: anthraquine-2-sulfonic acid sodium salt (AQMS), hexaaminoruthenium chloride (RuHex), a pair of potassium hexacyanoferrates (II) and (III) (KsFe (CN)e374') and a methylene blue (MB). Measurements were carried out using the SWV technique (the anode scan).

[0028] Fig. 6 shows a comparison of the current response for the disk gold electrodes modified with Al sequence (4 pM) and blocking agent being 6-mercapto-l -hexanol (4 pM), which were incubated with 100 nM manganese ions (in 50 mM MES, pH 6.0) in the presence of the redox indicators: anthraquinone-2-sulfonic acid sodium salt (AQMS), hexaaminoruthenium chloride (RuHex), pair of potassium hexacyanoferrates (II) and (III) (K3Fe(CN)e374') and methylene blue (MB). Measurements were carried out using the SWV technique (the cathode scan).

[0029] Fig. 7 shows a comparison of the current response for the gold disk electrodes modified with Al sequence at the specific concentrations and blocking agent being 6-mercapto-l -hexanol (4 pM), which were incubated with the blank (100 pM AQMS) and 100 nM manganese ions (in 50 mM MES, pH 6.0). Measurements were carried out using the SWV technique (the anode scan).

[0030] Fig- 8 shows a comparison of the current response for the gold disk electrodes modified with Al sequence at the specific concentrations and blocking agent being 6-mercapto-l -hexanol (4 pM), which were incubated with the blank (100 pM AQMS) and 100 nM manganese ions (in 50 mM MES, pH 6.0). Measurements were carried out using the SWV technique (the cathode scan).

[0031] Fig- 9 shows a comparison of the current response for the gold disk electrodes modified with Al sequence (4 pM) and blocking agent being 6-mercapto-l -hexanol at the specific concentrations, which were incubated with the blank (100 pM AQMS) and 100 nM manganese ions (in 50 mM MES, pH 6.0). Measurements were carried out using the SWV technique (the anode scan).

[0032] Fig. 10 shows a comparison of the current response for the gold disk electrodes modified with Al sequence (4 pM) and blocking agent being 6-mercapto-l -hexanol at the specific concentrations, which were incubated with the blank (100 pM AQMS) and 100 nM manganese ions (in 50 mM MES, pH 6.0). Measurements were carried out using the SWV technique (the cathode scan).

[0033] Fig- 11 shows a calibration curve for the gold disk electrodes modified with Al sequence (4 pM) and blocking agent being 6-mercapto-l -hexanol (4 pM). Measurements were carried out using the SWV technique (the cathode scan) in the presence of the redox indicator (100 pM AQMS).

[0034] Fig. 12 shows a comparison of selectivity for the gold disk electrodes modified with Al sequence (4 pM) and blocking agent being 6-mercapto-l -hexanol (4 pM). Measurements were carried out using the SWV technique (the cathode scan) in the presence of the redox indicator (100 pM AQMS). Fig. 13 shows a comparison of the current response for the gold disk electrodes and the miniature from BVT Technologies modified with Al sequence (4 pM) and blocking agent being 6-mercapto-l -hexanol (4 pM) in MES buffer (50 mM, pH 6.0) for a manganese ion concentration of 250 nM. Measurements were carried out using the SWV technique (the cathode scan) in the presence of the redox indicator (100 pM AQMS).

[0035] Fig. 14 shows a comparison of the current response for the gold disk electrodes modified with Al sequence (4 pM) and blocking agent being 6-mercapto-l -hexanol (4 pM) in MES buffer and in the real sample (tap water) for a manganese ion concentration of 250 nM. Measurements were carried out using the SWV technique (the cathode scan) in the presence of the redox indicator (100 pM AQMS).

[0036] Fig. 15 shows a comparison of the current response for the gold disk electrodes modified with Al sequence (4 pM) and blocking agent being 6-mercapto-l -hexanol (4 pM) in MES buffer and in MEM medium sample without and with an addition of 10 nM manganese ions. Measurements were carried out using the SWV technique (the cathode scan) in the presence of the redox indicator (100 pM AQMS).

[0037] Examples of realisation

[0038] Example 1

[0039] A method of identification of an optimal aptamer sequence which comprises DNA oligonucleotides specific towards manganese ions (Mn2+).

[0040] In order to identify an appropriate sequence, a unique VENNMultiplex™ SELEX technique was used due to a small size of the detected analyte. The SELEX procedure used is based on a hybridization process of the identified aptamer sequences with complementary sequences that are immobilized on the surface of magnetic nanoparticles. The sequence identification procedure includes the use of the VENNMultiplex™ SELEX technique in 12 selection rounds, followed by a next-generation sequencing (NGS) and a bioinformatics research.

[0041] As obtaining a sufficient pool of enriched sequences (fcount number was below 10) additional rounds of selection were applied, which allowed to reach the pool of 3.6 million readouts, where 700000 constituted the pool of 73000 unique sequences. Thanks to the elimination of cross interactions it was possible to acquire so called fcount number of 173, which indicates acquiring a set of sequences that have a specific selectivity towards manganese ions. Subsequently, values of delta G parameter that determines sequence structural stability were compared which allowed to identify 8 DNA aptamer sequences of 32 nucleotides in length and finally 1 DNA aptamer sequence (Al sequence) of 32 nucleotides in length was selected.

[0042] The aptamer Al sequence, after its identification, was synthesized.

[0043] Example 2

[0044] The use of the aptamer as a receptor layer of an aptasensor for detection of manganese ions.

[0045] Electrochemical measurements were carried out with use of miniature electrodes system comprising two gold working electrodes modified with Al sequence (electrode no. 1) as well as with polyT reference sequence (electrode no. 2), a reference electrode and an auxiliary electrode.

[0046] To Al sequence 5’-TAG CGC CGA AGA ACG ACA ATT TTA GAT CGA CA - 3’ functional groups may be bonded for example, in the form of disulphide groups that allow among other for immobilization of the sequence on the surface of a transducer as well as molecules with electroactive or fluorescence properties that constitute a source of an electrochemical or optical response when the sequence is used as the aptasensor receptor layer. It is possible to additionally reduce disulphide groups and obtaining of thiol groups, which favours an increase of a packing density of the receptor layers on the surface of gold. However, it is not essential for formation of the sensing layer, moreover too dense packing of the receptor layer may decrease its flexibility and capability to reorganize when interacting with the selected analyte.

[0047] In order to verify the usage possibilities of the aptamer sequence as the receptor layer of the aptasensor for the electrochemical detection of manganese ions, a commercially available miniaturised sensors “Dropsense” from Methrom company were used. 2 gold working electrodes were placed in the sensors, among which one was modified with the aptamer sequence (Al), and the second one with reference sequence polyT that comprised 10 thymine nucleotides, what was aimed to minimize a background signal and a current signal displacement. The aptamer sequence and the reference sequence comprised disulphide group at 5’ terminals, which allowed for a covalent bonding to the surface of gold.

[0048] Initially, the gold electrodes were sonicated in 100% ethanol for 480 s in two repetitions. Then the electrodes were subjected to an electrochemical scanning in 0.5 M NaOH solution with an electric potential in the range of -0.35 to -1.35 V at scanning rate of 1 V / s and with 50 scans with two repetitions. A following step was carrying out amperometric measurements in 0.5 M H2SO4 with the electric potential of 2 V for 5 s and -0.35 V for 10 s. Then, the electrochemical scanning was performed using a cyclic voltammetry technique in 0.5 M H2SO4 at the scan rate of 1 V / s (10 scans) and 0.1 V / s (4 scans) for the electric potential in the range of -0.35 to 1.5 V. The next step was an electrochemical scanning using the cyclic voltammetry technique in a solution of 0.01 M KC1 / 0.1 M H2SO4 in the electric potential range of -0.2 to 0.75 V (scan rate of 0.1 V / s 5 scans), 0.2 - IV (scan rate of 0.1 V / s, 5 scans), 0.2 - 1.25V (scan rate of 0.1 V / s, 5 scans), 0.2 - 1.5V (scan rate of 0.1 V / s, 5 scans). The last step of preparation of the surface of the gold electrodes was an electrochemical scanning using the cyclic voltammetry technique in a 0.05 M H2SO4 solution using the electric potential in the range from -0.35 to 1.5 V at a scan rate of 0.1 V / s (6 scans with two repetitions). The disulphide group of the Al sequence was reduced by mixing 1 pL of 100 pM of the sequence with 1 pL of 20 mM TCEP and incubation at a room temperature for 1 h.

[0049] The formation of the receptor layer was carried out by an overnight incubation of the gold electrodes in 500 nM aptamer sequence / poly T solution, which had previously been heated for 5 min at 95 °C and cooled to a room temperature for 15 min, followed by rinsing the electrodes in a buffer for folding the sequences (folding buffer) performed in triplicate, which was supplemented with an incubation for 1 h with a 2 mM solution of 6-mercapto-l- hexanol (MCH) filler in order to eliminate non-specific interactions on the surface of the electrode. Electrochemical measurements were carried out using the square-wave voltammetry (SWV) technique in the presence of a indicator "in the solution" - 10 mM potassium hexacyanoferrate (III) (K3Fe(CN)e) and the measurement was carried out using electrical potential ranging from -0.2 to 0.7 V for 50 Hz frequency. Measurements were first carried out in a solution of the redox indicator only, then in a solution of 100 nM manganese ions. The linear response range and the lower detection limit were determined for the electrodes modified with Al aptamer sequence. The dynamic response was recorded in the range from 10 pM to 100 nM (Fig. 3), however, dependence of the current response on the concentration of manganese ions was characterised by high standard deviations.

[0050] In next step a selectivity testing was carried out considering comparison of the responses of the electrodes that were incubated in manganese ions as well as in the mixture of interferring ions such as: Pb2+, Cd2+, Cu2+, Hg2+, UO22+(Fig.4). Due to a low repeatability of the electrodes’ response, the gold disk electrodes were used as transducers.

[0051] In order to prepare the receptor layers on the disk electrodes, the gold surface was cleaned beforehand using the following procedure. The disk electrodes were initially polished using aluminium oxide powder (AI2O3) with a grain diameter of 0.3 and 0.05 pm for about 2 min and then were sonicated in a solution of ethanol and water (1 : 1 (v / v)). The next step was 1 min incubation of the electrodes in a piranha solution (hydrogen peroxide: sulphuric acid (JLCh ThSC ), 1 :3, (v / v)), followed by an electrochemical scanning using the cyclic voltammetry (CV) in the potential range -0.3 - 1.7 V for a scanrate of 0.3 V / s, 10 cycles. Confirmation of obtaining a clean gold surface was determined on the basis of graphs recorded by cyclic voltammetry in 5 mM K3Fe(CN)e / K4Fe(CN)6 solution for an electric potential range from -0.2 to 0.6 V, at a scan rate of 0.1 V / s, 2 cycles.

[0052] The electrode modification was carried out by incubation for 2 h in 4 pM aptamer solution, followed by 1 h incubation in 4 pM 6-mercapto-l -hexanol filler solution (in 1 M KH2PO4, pH 4.5 buffer) (Fig.1). During the electrochemical measurements, as a basic solution 50 mM MES of pH 6.0 was used that should not be undergoing a complexation with manganese ions.

[0053] Electrochemical studies aimed at confirming the possibility of using the aptamer sequence as the receptor layer for detection of manganese ions started with selecting the type of redox indicator that constituted the source of the current response in the sensor system. The following indicator were used in the measurements: methylene blue (50 pM), potassium hexacyanoferrate (II) / (III) pair (5 mM), anthraquinone-2-sulfonic acid sodium salt (AQMS) (100 pM) as well as hexaaminoruthenium chloride (100 pM). The electrodes were initially incubated for 5 min in a solution of the selected indicator, after words the electrochemical measurements were carried out using cyclic voltammetry (CV) and square-wave voltammetry (SWV) techniques - considering the anode and the cathode scan. In the following step, 5 minutes incubation of the electrode working in the solution containing a specific redox indicator and enriched with manganese ions at a concentration of 100 nM was carried out. After this step, the electrode was placed back into the solution containing solely the redox indicator , and then voltammetric measurements were performed again. This made it possible to compare the response of the electrodes before and after the incubation with manganese ions and, as a result, to define the influence of Mn2+ions on the response of the sensor modified by the aptamers. The comparison also considered the response for the blank sample, which constituted MES solution containing the selected redox indicator only. Due to differences in the values of the electrochemical surface of the electrodes for the individual specimens, the aptasensors response was assumed to be a differential current signal recorded using the square-wave voltammetry (SWV) technique using the following equation: where Io indicates current signal recorded before, while I indicate current signal recorded after incubation of the electrodes in manganese ions.

[0054] The current responses in the presence of the individual redox indicators are shown in Fig. 5 and Fig. 6, which indicate that the largest changes in the current response were recorded during measurements using AQMS indicators, pair of potassium hexacyanoferrates (II) and (III) as well as methylene blue. In the presence of hexaaminoruthenium chloride (RuHex), there were no visible differences between the electrodes incubated in manganese ion solution and the blank sample. In turn, changes in the responses using methylene blue indicator did not exceed 0.05. It should be emphasized that the use of the pair of potassium hexacyanoferrates (II) and (III) as the redox indicator is not possible since after adding manganese salt indicator to the solution, precipitation of hexacyanomanganese complex occurs and the solution becomes cloudy (Adamson, A. W. and al., 1950). Therefore, AQMS indicator was used in subsequent electrochemical measurements (Fig. 2).

[0055] Further tests allowed to match conditions of the receptor layer formation which considered the concentration of the aptamer (4 pM) (Fig.7 and Fig. 8) as well as of the blocking agent - MCH (4 pM) (Fig. 9 and Fig. 10). Subsequently the linear response of the aptasensor was determined, which was in the range from 50 nM to 1 pM (Fig.11). Based on the figure representing the dependency of current response versus manganese ions concentration (Fig.11) an affinity constant Kd of the receptor layer to manganese ions was determined. For this purpose, the following equation was used: where: S is aptasensor response , Smax is maximum aptasensor response, [Mn2+] is manganese ions concentration.

[0056] The affinity constant Kd was 64 nM which indicates strong bonding of manganese ions with the receptor layer formed by the aptamers and particles of 6-mercapto-l -hexanol filler.

[0057] Further step was determination of the selectivity of the aptamer receptor layer by comparison of current responses after incubation of the electrodes in solution comprising 250 nM manganese ions and the interfering ion at the same concentration (Fig.12). The highest response was achieved for Mn2+ions, which was two times higher than the response for uranyl (UO22+), nickel (Ni2+) or antimony ions. In case of cadmium (Cd2+), mercury (Hg2+) and chromium (CrC2') ions a change of response was observed that indicated an increase of the current signal after incubation of the electrodes with abovementioned cations. In case of mercury ions, a different characteristic of the response may be justified by presence of 6 thymine bases, which most likely cause the formation of dithymine bridges and changes of an aptamer conformation, resulting in an increased level of the affinity of AQMS molecules to the electrode surface. On the other hand, the conformation changes in case of the interaction of the aptamers with cadmium ions, may be explained by the presence of thymine and guanine bases containing oxygen and nitrogen atoms, which may form strong interactions with Cd2+ions. An accepted limit of the manganese ions concentration according to the WHO guidelines (1.82 pM) and the Polish guidelines (0.91 pM). For that reason, responses of the aptasensor for manganese ions and the interfering ions of 250 nM, which is the concentration below the maximum allowed concentration and at the same time is in the range of the linear response of the aptasensor, was chosen for comparison

[0058] In the further step the possibility to use commercial miniature sensors, from BVT Technologies company, as transducer for fabrication the aptamer receptor layers were checked. The aptamer layer was made in the same manner as when using the disk electrodes and the same measurement procedure was used. The response obtained for the miniature converters incubated in 250 nM solution of manganese ions is slightly higher than the response for the disk electrodes, but is distinguished by the significant standard deviation (Fig. 13). The possible cause is the diversity in the surface density of the aptamers on the surface of the screen-printed transducers, which affects the final response of the sensors.

[0059] Example 3

[0060] The use of the aptasensor for detection of manganese ions in water samples and other liquid samples.

[0061] The last step of the tests was the verification of the usability of the aptasensor by comparing the response for the laboratory sample of manganese ions prepared in MES buffer and the sample of tap water enriched with manganese ions. The laboratory sample constituted a solution of 50 mM MES (2-(N-morpholino)ethanesulfonic acid) with pH of 6.0 with addition of 100 pM of AQMS redox indicators as well as containing manganese ions at a concentration of 250 nM. In turn, to prepare the sample of tap water, 1 mL of tap water was used, in which a specific weighed amount of manganese nitrate was dissolved, what made possible to prepare 10 mM solution of manganese ions. The sample was then diluted to a concentration of 250 nM Mn2+ions by using 50 mM MES (2-(N-morpholino)ethanesulfonic acid) solution of pH 6.0 with addition of 100 pM AQMS redox indicator. The procedure of cleaning the gold electrodes as well as their modification with the aptamer sequence and the blocking agent being 6-mercapto-l -hexanol was identical to the procedure described above. The electrochemical measurement consisted of incubating the modified electrodes for 5 min in 100 pM AQMS solution, subsequently electrochemical measurements were carried out using cyclic voltammetry (CV) and square-wave voltammetry (SWV) techniques - including the anode and the cathode scanning. In the next step, 5 min incubation of the working electrode in the solution of the laboratory sample or the sample of the tap water enriched with manganese ions were carried out. After this step, the electrode was placed back into the solution containing the redox indicator only, and then voltammetric measurements were performed again. The responses for the electrodes incubated in MES buffer and the tap water sample are almost identical and the aptasensor response is at the level of 0.16. Based on the results obtained, it can be concluded that matrix components in the form of the tap water have a minimal impact on the aptasensor response. At the same time, the change in the current response recorded in the case of the analysis of the tap water sample was caused by the enrichment of the water sample with manganese ions at a concentration of 250 nM. This confirms the possibility of using the gold disk electrodes modified with the aptamer sequence for the analysis of the real samples in the form of the tap water (Fig. 14).

[0062] Due to the possible presence of manganese ions in the other liquid samples for example in the medium intended for cell culture, an analysis of selected media was also carried out, which were subjected to lOx dilution with 50 mM MES (2-(N-morpholino)ethanesulfonic acid) solution of pH 6.0 with addition of 100 pM redox indicator AQMS. One sample constituted a diluted solution of the medium, while the other sample of a diluted solution of the medium was enriched with manganese ions of 10 nM concentration. Manganese ions enriched sample was prepared by dissolving a determined weighed amount of manganese nitrate in the 10 times diluted medium for cell culture, which resulted in obtaining an initial concentration of 10 mM. The solution prepared in such a way was then diluted to a final concentration of 10 nM of manganese ions. The procedure of cleaning of gold electrodes as well as their modifications with the aptamer sequence and the blocking agent being 6- mercapto-1 -hexanol, was identical to the procedure described above. The electrochemical measurement was performed by incubation of the modified electrode for 5 min in 100 pM AQMS solution, and subsequently the electrochemical measurements were carried out using cyclic voltammetry (CV) and square wave voltammetry (SWV) techniques - including the anode and the cathode scan. In the next step 5 min incubation of the working electrode in the solution of the diluted medium or the diluted medium enriched with manganese ions were carried out. After this step, the electrode was placed back into the solution containing redox indicator only, and then voltammetric measurements were performed again. A comparison of the current response for the electrodes incubated in MES buffer, MES buffer with addition of 10 nM manganese ions, the diluted MEM medium from VWR company and the diluted MEM medium from VWR company with addition of 10 nM manganese ions indicated that the response of the electrodes modified with the aptamer sequence for the sample of the medium for the cell culture enriched with manganese ions is slightly higher (0.07) than in the case of the laboratory sample of MES buffer enriched with manganese ions (0.04) after taking into account the response for MES buffer and the diluted MEM medium without the addition of manganese ions. The slightly higher response in case of the sample of the medium for cell culture enriched with manganese ions may result from the complexity of the analysed sample of the culture medium, the components of which could have influenced the final response of the aptasensor. Additionally, based on the results obtained, the presence of manganese ions in the MEM medium from VWR company (Fig. 15) can be concluded.

[0063] List of reference signs

[0064] 1 - gold electrode

[0065] 2 - receptor layer

[0066] 3 - aptamer

[0067] 4 - filler

[0068] 5 - anionic indicator

[0069] List of Publications:

[0070] 1. M. Mizunuma, M. Suzuki, T. Kobayashi, Y. Hara, A. Kaneko, K. Furukawa, Y. Chuman, Development of Mn2+ - Specific Biosensor Using G-Quadruplex-Based DNA, Int. J. Mol. Sci. 2023, 24, 11556.

[0071] 2. A. W. Adamson, J. P. Welker, M. Volpe, Exchange Studies with Complex Ions .1. The Exchange of Radiocyanide with Certain Heavy Metal Complex Cyanides, J Am Chem Soc, 1950, 72, 4030- 4036.

[0072] List of abbreviations:

[0073] AQMS - anthraquinone-2-sulfonic acid sodium salt

[0074] AES - atomic absorption spectrometry

[0075] CV - cyclic voltammetry

[0076] DNA - deoxyribonucleic acid

[0077] EDXRF - energy dispersive X-ray fluorescence spectrometry

[0078] ICP - AES - atomic absorption spectrometry coupled with microwave plasma

[0079] MB - methylene blue

[0080] MCH - 6-mercapto-l -hexanol

[0081] MEM - minimum essential medium

[0082] MES - 2-(N-morpholino)ethanesulfonic acid

[0083] NGS - next generation sequencing

[0084] PCR - polymerase chain reaction

[0085] RuHex - hexaaminoruthenium

[0086] SELEX - systematic evolution of ligands through exponential enrichment

[0087] SWV - square wave voltammetry Sequence listing ST 25

[0088] <110> Politechnika Wars zawska

[0089] <120> Aptamer, aptasensor comprising aptamer, and use of the aptamer for selective detection of manganese ions

[0090] <130> 60340

[0091] <160> 1

[0092] <170> BiSSAP 1 . 3 . 6

[0093] <210> 1

[0094] <211> 32

[0095] <212> DNA

[0096] <213> Arti ficial Sequence

[0097] <220>

[0098] <223> Aptamer for detection of manganese ions

[0099] <400> 1 tagcgccgaa gaacgacaat tttagatcga ca 32

[0100] Sequence listing ST 26

Claims

Claims1. An aptamer capable of specific detection of manganese ions characterised, characterised in that, has a nucleotide sequence as shown in SEQ. ID NO.l2. The aptamer according to claim 1, characterised in that, is modified with a disulfide group.

3. The aptamer according to claim 1 characterised, characterised in that, the disulfide group modification is on the 5’ terminal.

4. An aptasensor having an affinity to manganese ions Mn2+, characterised characterised in that, comprises a gold working electrode (1) coated with a receptor layer (2) that comprises aptamers (3) as described in claim 1, and filler (4) molecules as well as an anionic indicator (5) that is present in the solution.

5. The aptasensor according to claim 4, characterised in that, is an electrochemical sensor.

6. The aptasensor according to claim 4, characterised in that, the receptor layer (2) is covalently bonded to a surface of the electrode.

7. The aptasensor according to claim 4, characterised in that, the filler (4) is 6-mercapto-l- hexanol.

8. The aptasensor according to claim 4, characterised in that, the anionic indicator (5) for generating a current signal is anthraquinone-2-sulfonic acid sodium salt.

9. The aptasensor according to any one of claims from 4 to 8, characterised in that, the affinity of the receptor layer (2) to manganese ions is 64 nM.

10. The aptasensor according to any of claims from 4 to 9, characterised in that, the range of its dynamic linear response is from of 50 nM to 1 pM.

11. A method of obtaining an aptasensor according to claim 5 comprising the steps of: a) formation of a receptor layer (2) by incubation of the gold electrodes (1) in aptamer (3) solution for 2 h, b) incubation with a filler (4) solution for 1 h to eliminate nonspecific interactions at the surface of the electrode.

12. The method according to claim 11, characterised, characterised in that, the concentration of the solution from step a) is 4 pM.

13. The method according to claim 11, characterised, characterised in that, the filler (4) is 6- mercapto- 1 -hexanol .

14. The method according to claim 11, characterised in that, the concentration of the filler (4) is 4 pM.

15. The use of the aptamers according to claim 1 as a receptor layer of a biosensor for in situ selective detection of manganese ions by electrochemical techniques.

16. The use according to claim 15, characterised in that, manganese ions are detected in liquid samples.

17. The use according to claim 15, characterised in that, manganese ions are detected in water.

18. The use according to claim 15, characterised in that, manganese ions are detected in a culture medium.

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