The subject of the invention is a buffer solution and its use in electrochemical impedance spectroscopy to detect pathogenic bacteria and viruses.

Rapid detection and identification of the pathogen responsible for the infection is a major aim and challenge for diagnostic centers. In recent years, the classical methods of pathogen detection, such as bacterial culture and identification using phenotypic biochemical tests or plaque forming cells test for viruses, have been supplemented with modern analytical and molecular methods. Among these methods, Electrochemical Impedance Spectroscopy (EIS) can be distinguished.

Electrochemical impedance spectroscopy involves the measurement of impedance between the operating and auxiliary electrodes in the widest possible frequency range from ImHz to 100 MHz. The EIS method is used to determine the speed of electrode reactions and the characteristics of the electrode-electrolyte solution phase limit. It also allows for indirect assessment of the surface structure of the electrode. Electrochemical impedance spectroscopy also allows for precise detection of pathogens.

The principle of the EIS is to determine the impedance of an electrochemical sensor by application of a small (typically several to several dozen millivolts) sinusoidal voltage of specific frequency (typically 1 mHz to 1 MHz) to the sensor electrodes and measurement of the current flowing through the circuit. Additionally, electrochemical sensors are polarized with DC voltage typically ranging from a few to several hundred millivolts, whose purpose is to reduce the non-linearity of electrochemical sensor characteristics or to generate conditions necessary for the occurrence of chemical reactions that are essential for the operation of the sensor.

The impedance of an electrochemical sensor is frequency-dependent and can be expressed as follows as a complex number:

Z(f) = Z0 (coscp + i sincp) where f is frequency, Z0 is impedance module and f is phase shift between current and voltage. The Z(f) complex impedance consists of the real and the imaginary part. The graph where the imaginary part of the impedance is plotted against the real part (the so-called "Nyquist diagram") for different frequencies is commonly used to visualize the impedance characteristics of electrochemical cells and sensors.

The EIS method uses impedimetric biosensors. When a target substance, such as a protein, binds to receptor molecules previously bound to the electrode surface of the impedance biosensor, the impedance value of the sensor changes. The difference in impedance measured before and after binding the target substance to the receptor molecules allows for the detection of presence of the target substance in the solution.

In the current state of the art, certain ways of detecting selected pathogens are known. They are usually optical means such as microscopy or fluorescence. As a standard, antibodies recognizing selected biomarkers are used to detect pathogens. Another way is the use of aptamers, nucleic acid fragments or antibody fragments or peptides. In case of the latter, there are difficulties with constructing such a molecule that will be firmly attached to the support and will selectively recognize the selected pathogens.

Another challenge is the simultaneous recognition of viruses and bacteria in single sample. This is particularly important in case of diagnostics of infections where co-infections occur. In case of upper respiratory tract infection, co-infections with bacteria of the genus Streptococcus (e.g. S. pyogenes, S. pneumoniae ), Haemophilus influenzae or Rhinovirus are often associated with infection with influenza virus. Correct detection of occurring pathogens allows for the selection of appropriate treatment methods, faster recovery, and in effect there is no need to use antibiotics when exclusively viruses are the source of infection.

Among the techniques that allow for precise detection of a whole range of pathogens, the most effective methods are molecular (RT-PCR) or immunoenzymatic (ELISA). Both of these methods are sensitive and specific, but require specialist knowledge, professional equipment, reagents and a laboratory. They are also expensive and time consuming, which is why there is search for alternative methods, that would be equally reliable, but easier to use, cheap and quick.

Electrochemical impedance spectroscopy is becoming increasingly popular in pathogen detection. There are a number of examples in scientific literature describing the use of EIS for pathogen identification. The publication by Malvano et al (Sensors (Basel)) 2018 Jul; 18(7): 2168) describes the use of EIS to detect pathogens in food. A method for detection of Escherichia coli 0157:H7 using antibody based sensors has been developed. PBS buffer containing K ₃ [Fe(CN) ₆ ]/K ₄ [Fe(CN) ₆ ] was used for detection, in the ratio 1:1 with pH=7. Whereas, Louie et al. (Field Analyt Chem Technol 2: 371-377, 1998) described a method based on the measurement of impedance changes for the detection of E. coli and Salmonella spp. in food. This is an example of detecting more than one pathogenic strain of bacteria in a sample, but it cannot be used in combined bacterial-viral infections. Standard water buffers were used to detect pathogens, without addition of detergent that, when used in excess, interferes with the course of electrode reactions and additionally causes a strong drop in the buffer/electrolyte surface voltage, so it can negatively affect the measured EIS parameters.

Document AU2011275591B2 reveals how microorganisms can be detected by providing growth-friendly conditions for growth of microorganisms in suitable containers, and then different methods are used to identify these strains. The advantage of the invention are shortened detection time, ease-of-use and the ability to detect multiple pathogens simultaneously. One of the detection methods can be electrochemical impedance spectroscopy. The invention shortens the time needed to identify pathogens, but only when it is compared to cultures that usually last several days. The essence of this invention is to create appropriate conditions for the growth of microorganisms, which requires time (at least several hours). In addition, it is not possible to detect viruses with this method, which eliminates it for use in bacterial-viral co-infection detection.

US patent US6743581B1 describes how to detect analytes such as DNA fragments, RNA, proteins, peptides and microorganisms. The described method is a complicated platform that consists of bio-chips modified with antibodies or molecular material and detection system based on Complementary Metal-Oxide Semiconductor (CMOS) technology. The system itself is expensive and complicated in production. Also, the preparation of the sample for analysis is dependent on the type of analyte, which increases the time with the costs related to the diagnostic process.

Japanese patent application JP2002518998A deals with the electrochemical method of detecting multiple nucleotide sequences in single sample. The system consists of multiple microelectrodes covered with DNA chains that recognize selected nucleotide sequences in the samples. The advantage of this method is that it is not necessary to multiply the genetic material. The disadvantage is that only DNA can be detected, which excludes the possibility of recognizing viruses based on RNA as genetic material, as in the case of the influenza virus. In addition, the kit used for diagnostics based on the detection of genetic material usually requires adequate sample preparation, which takes time and constitutes extra costs. The genetic material is susceptible to contamination and degrades rapidly if it is not properly secured.

Yu et al. (Analyst 2006 Jun;131(6):745-50) described an impedance biosensor based on a matrix to study multiple antigen-antibody interactions simultaneously. A buffer of 5 mM [Fe(CN) ₆ ] ^{3 /4} in 10 mM PBS was used for analysis. The system is effective for proteins and antigen detection. No effects on whole microorganisms have been shown, therefore, the method is not suitable for quick diagnostics without prior sample preparation.

The addition of further substances to the measurement buffer is undesirable because it disturbs the reaction environment, so it is difficult to build a universal sensor. Moreover, the addition of detergent to the measurement buffer decreases the reaction sensitivity. Detergent molecules surround the protein and block the flow of ions through the system, that is why measuring the effect of proteins as analytes with EIS is done in a detergent-free buffer.

Electrochemical impedance spectroscopy can be an effective tool for rapid identification of biomarkers in samples. However, this method has its limitations. One of them is the need to apply the redox system in the reaction buffer. The environment of the reaction itself together with the redox system must be strictly defined, therefore the addition of other substances such as salt ions, proteins, detergents is undesirable. These limitations make the EIS method is currently not suitable for all analyses, including analyses of genetic material or wide range of microorganisms. The role of gold standards is still played by molecular (PCR) or immunoenzymatic (ELISA) methods.

PCR is a method for detecting genetic material. The advantage of this method is its high sensitivity and specificity. However, its wide application in diagnostics is limited by high cost of reaction equipment, high cost of reagents, required high purity of the laboratory and presence of highly qualified personnel.

The immunoenzymatic method, in turn, is suitable for detecting protein biomarkers. It is cheaper than PCR, but less sensitive and also needs to be operated by highly qualified personel in a clean laboratory. These limitations make it necessary to look for other alternative methods allowing for rapid biomarker detection, methods that would be simple to use, have low costs and would be reliable. The pathogen detection methods present in the state of art do not allow for the simultaneous detection of multiple bacteria and viruses by the EIS method. This is due to the different structure of gram positive, gram negative bacteria and viruses. In order to make possible detection of pathogenic bacteria and viruses in single sample by electrochemical impedance spectroscopy, it is necessary to allow pathogens to interact with molecules (antibodies, aptamers, peptides and DNA) on the electrode surface, which is not guaranteed by the basic buffers/electrolytes commonly used in the art, necessary to record the occurring reactions on the electrode/near the electrode. In case of many sensors, pathogen diagnostics is performed by the detection of selected proteins or fragments of genetic material that is only characteristic for a given pathogen. In some cases these are proteins located in the membrane or cell wall of the microorganisms, in others it is necessary to break down the pathogen to release intracellular proteins. The above described method therefore does not allow for simple and rapid detection of pathogens in single sample during one measurement, which in some cases is necessary when the high risk of co-infection occurs.

An example is the detection of upper respiratory tract infections in humans, where in the winter season influenza often occurs along with bacterial co-infections. To detect an entire panel of pathogens, different methods are used, such as microbial culture for bacteria or PCR mainly for viruses. There is no method that can detect both types of pathogens simultaneously in one and the same sample obtained from the patient. EIS methods are described in the literature, but for the detection of bacteria only.

Impedance spectroscopy is a method that is becoming increasingly popular in diagnostics, because it allows for obtaining results in a short time. The difficulty in wide application of this method is to develop reaction conditions that would allow for the detection of multiple pathogens at the same time, especially bacteria by detecting surface proteins and viruses, thanks to the destruction of capsid, additionally without disturbing the flow of ions in the solution.

The aim of the invention was therefore to develop a buffer solution that would enable dissolving the sample and carrying out measurement of the impedance variation for detection many pathogenic bacteria and viruses simultaneously in a single sample.

The subject of the invention is a buffer solution containing: buffer, potassium hexacyanoferrate (II), potassium hexacyanoferrate (III), and pH-regulating agent, characterized in that it contains surfactant.

Preferably, the buffer is selected from the group comprising Tris-HCl, PBS and HEPES, and the buffer concentration is within the range of 50 - 100 mM.

Preferably, the concentration of potassium hexacyanoferrate (II) is in the range of 1 - 10 mM.

Also preferably, the concentration of potassium hexacyanoferrate (III) is in the range of 1 - 10 mM. The surfactant is preferably selected from a group comprising Tween and Triton X, more preferably the surfactant is selected from a group comprising Tween 20 and Triton X-100 and the surfactant concentration is 0.1 - 2 % vol.

Preferably, the pH-regulating agent is selected from the group comprising: HC1, H2SO4, H3PO4, NaOH, KOH, Mg(OH) _2.

Additionally, preferably the pH of the buffer solution according to the invention is in the range of 5 - 9, more preferably of 7 - 8.

Particularly preferably, the buffer solution according to the invention is characterized in that it contains 100 mM Tris-HCl, 6.2 mM potassium hexacyanoferrate (II), 6.2 mM potassium hexacyanoferrate (III), 0.1 % vol. Tween 20 and its pH is in the range of 7.75-7.85.

Another subject of the invention is the use of a buffer solution according to the invention in electrochemical impedance spectroscopy to detect one or more pathogenic bacteria and viruses in a single sample.

Preferably, detected pathogenic bacteria and viruses are selected from the group comprising: genus Streptococcus , genus Staphylococcus , family Orthomyxo viridae , family Picornaviridae, genus Haemophilus , family Herpesviridae , genus Mycoplasma , genus Bordetella , family M or axe l laceae , genus Pseudomonas , family Enter obacteriaceae, genus Proteus , genus Enterococcus , family Papillomaviridae , genus Ureaplasma , genus Treponema , genus Neisseria , genus Chlamydia , genus Acinetohacter , genus Gardnerella , genus Bacteroides, family Parvoviridae, family Paramyxoviridae, family Coronaviridae .

More preferably, detected pathogenic bacteria and viruses are selected from the group comprising: S. pyogenes, Infuenza B, Rhinovirus, H. influenzae, EBV, M. pneumoniae, B. pertussis, A. baumani, S. aureus MRS A, P. aeruginosa, E.coli ESBL CTXM-15, E.coli ESBL TEM-1, K. pneumoniae.

The advantage of the developed buffer solution is that it provides appropriate electrochemical conditions for electrode reactions and prepares pathogens for interaction with the electrode surface. It is characteristic that after collecting a swab it is not needed to perform additional actions to examine several pathogens at once, it is enough just to dissolve the sample in the buffer solution according to the invention and then apply a few drops to the sensor and measure impedance.

The advantageous features of the invention are illustrated by the following figures supporting information contained in the embodiments:

Fig. 1. The Nyquist diagram shows the interactions of the H1N1 influenza virus and S. pyogenes bacteria on a sensor modified with HSCH2(CH2)8CH2-CONH-HTIHGAQ and HSCH2(CH2)8CH2-CONH-MLPFRTD molecules. Continuous lines present impedance measurement for influenza virus, dashed lines for S. pyogenes bacteria. Blank means impedance measurement on unmodified electrode, incubation - impedance measurement on electrodes modified by appropriate molecules, reaction - measurement of impedance after application of a mixture of influenza virus and S. pyogenes. Fig. 2A and 2B. PCR reaction result confirming the presence of S. pyogenes and influenza virus in the sample.

Fig. 2A. Graph of the dependence of HEX dye fluorescence on the time of reaction allowing identification of the presence of type A influenza virus. Below the graph are Ct values for individual samples.

Fig. 2B. Graph of fluorescence relation between the SybrGreen dye and the time of reaction, allowing for detection of the presence of S. pyogenes. The graph below shows Ct values for individual samples.

Fig. 3. - negative control - Nyquist diagram illustrating the interaction of the mixture Haemophilus influenzae , Pseudomonas aeruginosa, Bordetella parapertussis and Streptococcus pneumoniae , on a sensor containing electrodes modified with HSCH ₂ (CH ₂ ) ₈ CH ₂ -CONH-HTfflGAQ and HSCH ₂ (CH ₂ ) ₈ CH ₂ -CONH-MLPFRTD molecules. Continuous lines mean measurement of impedance for the influenza virus, dashed lines for S. pyogenes. Blank means measurement of impedance on the unmodified electrode, incubation means measurement of impedance on electrodes modified with appropriate molecules, no reaction - impedance measurement after application of mixture Haemophilus influenzae, Pseudomonas aeruginosa, Bordetella parapertussis and Streptococcus pneumoniae.

Fig. 4. Nyquist diagram showing interactions of the H1N1 influenza virus and S. pyogenes bacteria on a sensor containing electrodes modified with HSCH ₂ (CH ₂ ) ₈ CH ₂ -CONH- HTfflGAQ and HSCH ₂ (CH ₂ ) ₈ CH ₂ -CONH-MLPFRTD molecules. Reaction conducted in a reference buffer. Continuous lines mean measuring impedance for the flu virus, dashed lines for S. pyogenes. Blank means measurement of impedance on unmodified electrode, incubation - impedance measurement on electrodes modified with appropriate molecules, reaction - measurement of impedance after application of the mixture of influenza virus and S. pyogenes bacteria. The result indicates no reaction for the influenza virus and a positive reaction for S. pyogenes bacteria.

Fig. 5. Scheme of modification of the gold surface, which is intended to be sensitive to the presence of protein D found in Haemophilus influenzae.

Fig. 6. Voltammetry diagram showing individual steps of the gold electrode Au modification.

Fig. 7. Nyquist diagram showing individual steps of the gold Au electrode modification.

Fig. 8. Nyquist diagram showing individual steps of the gold Au electrode modification and sensor interaction with a positive sample in the form of: A) protein D; B) Haemophilus influenza bacteria.

Fig. 9. Nyquist diagram showing interactions with negative samples in the form of bacteria, which do not contain on their surface protein specific for anti-protein D antibodies, i.e. A) Pseudomonas aeruginosa bacteria (P. aeruginosa ); B) Streptococcus pyogenes bacteria (S. pyogenes ); C) Streptococcus pneumonia bacteria (S. pneumoniae).

Fig. 10. Nyquist diagram from a swab examination of a patient with an upper respiratory tract infection. The dashed lines concern the detection of S. pyogenes bacteria, continuous - influenza virus, the dotted line refers to mucin protein detection as swab collection check. Examples

Example 1. Preparation of an electrode coated with HSCH2(CH2)sCH2-CONH- HTIHGAQ and HSCH2(CH ₂ )8CH2-CONH-MLPFRTD bioreceptor molecules

(a) Synthesis and purification of a HSCH2(CH2)8CH2-CONH-HTIHGAQ bioreceptor molecule

The peptides were synthesized manually, using the Solid-Phase Peptide Synthesis (SPPS), with using Fmoc/Bu ¹ procedure. The first protected amino acid derivative (Fmoc-Gln(Trt)-OH) was attached to the carrier at a dose of 1 mol _eq Fmoc-Gln(Trt)-OH / 1 g of resin. The synthesis consisted of twelve repeated steps of Fmoc protective group deprotection, a-amino group, rinsing and connecting to another protected amino acid derivative. During the deprotection step, Fmoc protection groups were removed with 20% piperidine solution in DMF. After each step of deprotection, as well as after each step of connection, completeness of the reaction was checked with the chloranil/Kaiser test. The bioreceptor molecule containing peptide with HTIHGAQ sequence was synthesized on 2.2'-chlorotrityl resin. In the last step a linker molecule was attached to create a functional molecule HSCFhlGFysCFh-COOH bioreceptor to the peptide with the use of the below described DIC/HOBt acylination protocol.

11-Mrcpt-COOH acid attachment, DIC/HOBt protocol. Reagents:

Preparation of the acylating mixture

In a 10 or 15 ml falcon tube, 11-mercaptoundecanoic acid was weighed in a quantity calculated by the formula: mmol ll-Mrcpt-COOH = 2 * Mmol 11-Mrcpt-COOH * LOAD * Wlz where m li-Mrcpt-cooH - weight of the derivative for weighing [mg]

Mmoi li-Mrcpt-cooH - molar mass of the derivative LOAD - degree of resin load M _{z -} mass of resin [g] HOBt was weighed into the falcon tube with the weighed derivative in the quantity calculated using the formula:

Tttl HOBt = 2 * Mmol HOBt * LOAD * Ttlz * 0.9

Then the weighed derivative and HOBt were dissolved in 8-10 ml DMF and to the obtained solution of the derivative and HOBt in DMF, DIC was added in the amount calculated by the formulae:

TtiDic = 2 * Mmowic * LOAD * rtiz where mHOBt - HOBt mass to be weighed [mg]

MmoiHOBt - HOBt molar mass mz - mass of resin [g] mDic - DIC mass [mg]

LOAD - degree of resin load MmoiDic - DIC molar mass VDIC - DIC volume [mΐ] dDic - DIC Density (=0.806)

A syringe containing resin was filled with all the above-described solution. The filled syringe was placed on a laboratory rocker and swayed for a minimum of 45 minutes at room temperature. After this time, the solution was removed from the syringe by maximum pressing down the piston of the syringe. Then the syringe containing the resin was filled with about 15 ml DMF, the syringe was again placed on the laboratory rocker and swayed for 2 minutes. The solution was again removed and the DMF syringe was filled twice. Then the syringe containing the resin was filled with about 15 ml DCM, the syringe was placed again on a laboratory cradle and swayed for 2 minutes. The solution was again removed and the DCM syringe filling was twice repeated. Then these steps were repeated three times with the use of about 15 ml DMF.

After the synthesis, the HSCH2(CH2)8CH2-CONH-HTIHGAQ bioreceptor molecule was detached from the resin, with simultaneous deprotection of protective groups of amino acid residues lateral functions, using a reaction mixture based on TFA (L-reagent) for up to two hours. The raw product, from the post-reaction mixture, was precipitated with cold diethylat ether (50ml) and lyophilized. The resulting raw bioreceptor molecule with the formula HSCH2(CH2)8CH2-CONH-HTIHGAQ was purified with high-performance liquid chromatography in a reversed phase system of the preparation column type C18 at the gradient between A and B, 20-80% B, where B is 100% acetonitrile (ACN) and A is 0.1% TFA in water. Eluates were fractionated and then analyzed by the RP-HPLC analytical method with linear gradient between A and B, 20-80% B, where B is 100% acetonitrile (ACN) and A is 0.1% TFA in water on analytical column type Cl 8. The fractions of the highest purity were combined and lyophilized.

Analogically, a bioreceptor molecule HSCH2(CH2)8CH2-CONH-MLPFRTD was synthesized using a protected derivative of Fmoc-Asp(OtBu)-OH attached to resin 2.2'-chlorotrityl and the HSCH ₂ (CH ₂ ) ₈ CH ₂ -COOH molecule.

(b) Modification of the electrode surface with a bioreceptor molecule obtained in step a)

The electrodes are cleaned before use with ethanol and ammonia-hydrogen peroxide mixture diluted with deionized water in a volume ratio of 1:1:18 respectively. Panels with electrodes were immersed in ethanol for 6 minutes, washed with deionized water and dried in an inert gas stream - argon.

Solutions of HSCH2(CH2)8CH2-CONH-HTIHGAQ bioreceptor molecules and HSCH2(CH2)8CH2-CONH-MLPFRTD bioreceptor molecules are applied to the cleaned gold surface, respectively 4 fields with one molecule, 4 fields with the other.

The cleaned gold surface was treated with 2 mΐ of peptide solution modified with thiol group. The peptide sequence (HTIHGAQ) is specific for the protein on the surface of Streptococcus pyogenes bacteria. The peptide was dissolved in a mixture of acetonitrile and deionized water in a volume ratio 2:13 (ACN:WDI) to the concentration 2.078· 10 ⁴ M. The obtained peptide solution was diluted with deionized water to the concentration of 1.75· 10 ⁵ M. The sensors were left in a dark place with 100% humidity, temperature of 5 - 6 ° C for 22-26 h. Then the electrode surface was washed with deionized water and dried in an inert gas stream.

The electrode, prepared in such way, covered with bioreceptor molecules is prepared for EIS measurements.

Example 2. Preparation of buffer solution (Tris buffer, Tween 20 surfactant)

On an analytical scale 1.2114 g of Tris buffer, 0.261 g of potassium hexacyanoferrate (II) and 0.204 g of potassium hexacyanoferrate (III) were weighed. The weighed amounts were dissolved in 80 ml of deionized water. Then 100 mΐ of Tween 20 surfactant was added and mixed on a magnetic stirrer. Diluted HC1 acid was added dropwise until pH 7.75 was obtained, then adjusted to 100 ml with deionized water.

Example 3. Preparation of buffer solution (Tris buffer, Surfactant Triton X-100)

On an analytical scale 1.2114 g of Tris buffer, 0.261 g of potassium hexacyanoferrate (II) and 0,204 g of potassium hexacyanoferrate (III) were weighed. The weighed amounts were dissolved in 80 ml of deionized water. Then 100 mΐ of Triton X-100 detergent was added and mixed on a magnetic stirrer. Diluted HC1 acid was added dropwise until pH 7.75 was obtained, then adjusted to 100 ml with deionized water.

Example 4. Preparation of buffer solution (100 mM PBS buffer, Tween20 surfactant)

On an analytical scale, 8 g of NaCl salt, 0.2 g of KC1 salt, 1.44 g of Na ₂ HP0 ₄ salt, 0.24 g of KH2PO4 salt, 0.261 g of potassium hexacyanoferrate (II) and 0.204 g of potassium hexacyanoferrate (III) were weighed. The weighed amounts were dissolved in 80 ml of deionized water. Then 100 mΐ of Tween 20 surfactant was added and mixed on a magnetic stirrer. Diluted HC1 acid was added dropwise until pH 7.4 was obtained, then adjusted to 100 ml with deionized water.

Example 5. Preparation of buffer solution (100 mM PBS buffer, Triton X-100 surfactant)

On an analytical scale, 8 g of NaCl salt, 0.2 g of KC1 salt, 1.44 g of Na ₂ HP0 ₄ salt, 0.24 g of KH2PO4 salt, 0.261 g of potassium hexacyanoferrate (II) and 0.204 g of potassium hexacyanoferrate (III) were weighed. The weighed amounts were dissolved in 80 ml of deionized water. Then 100 mΐ of Triton X-100 surfactant was added and mixed on a magnetic stirrer. Diluted HC1 acid was added dropwise until pH 7.4 was obtained, then adjusted to 100 ml with deionized water.

Example 6. Preparation of buffer solution (100 mM HEPES buffer, Tween 20 surfactant)

On an analytical scale, 2.38 g of HEPES buffer, 0.261 g of potassium hexacyanoferrate (II) and 0.204 g of potassium hexacyanoferrate (III) were weighed. The weighed amounts were dissolved in 80 ml of deionized water. Then 100 mΐ of Tween 20 surfactant was added and mixed on a magnetic stirrer. Diluted NaOH base was added dropwise until pH 7.4 was obtained, then adjusted to 100 ml with deionized water.

Example 7. Preparation of buffer solution (100 mM HEPES buffer, Triton X-100 surfactant)

On an analytical scale, 2.38 g of HEPES buffer, 0.261 g of potassium hexacyanoferrate (II) and

0.204 g of potassium hexacyanoferrate (III) were weighed. The weighed amounts were dissolved in 80 ml of deionized water. Then 100 mΐ of Triton X-100 surfactant was added and mixed on a magnetic stirrer. Diluted NaOH base was added dropwise until pH 7.4 was obtained, then adjusted to 100 ml with deionized water.

Example 8. Preparation of buffer from the publication of Malvano et al (Sensors (Basel)) 2018 Jul;18(7): 2168, e.g. 1 mM solution of potassium hexacyanoferrate (II)/(III) in PBS, 0.1 M with pH 7 - reference buffer

To prepare 0.1 M of the PBS buffer solution with a pH of 7.4 on an analytical scale, 10.988 g of Na ₂ HP0 ₄ salt (sodium hydrogen phosphate), 2.712 g of anhydrous NaH ₂ P0 ₄ salt (sodium dihydrogen phosphate) and 0.746 g of KC1 salt (sodium chloride) were weighed.. The weighed amounts were dissolved in 100 ml of deionized water. Then 0.0329 g of K ₃ [Fe(CN) ₆ ] salt (potassium hexacyanoferrate (III)) and 0.0368 g of K ₄ [Fe(CN) ₆ ] salt (potassium hexacyanoferrate(II)) were weighed on an analytical scale. The weighed amounts were dissolved in 100 ml of previously prepared PBS buffer to obtain 1 mM solution of potassium hexacyanoferrate (II)/ (III) in PBS, 0.1 M at pH 7.

Example 9. Use of a buffer solution according to the invention in an electrochemical impedance spectroscopy for the detection of influenza virus and Streptococcus pyogenes bacteria

The measurement buffer was prepared according to example 2. The electrodes were prepared according to example lb. To detect the presence of the influenza virus and Streptococcus pyogenes bacteria in single sample the following measurement protocol was followed.

The positive test is a mixture of an H1N1 virus, (ATCC A/PR/8/34) suspended in PBC buffer, titre 10 ⁷ CEID o/mL and S. pyogenes bacteria suspended in TBS buffer OD ₆ oo=TO.

A sensor, containing electrodes modified by molecules recognizing influenza and S. pyogenes was placed in the HDMI edge connector and connected to the potentiostat containing an FRA card for impedance measurements (Autolab M204).

Approximately 150 mΐ of measurement buffer obtained from example 2 was applied to the electrode surface.

The first step of measurement has commenced - calibration of the electrode. 150 mΐ of the measurement buffer obtained according to example 2 was applied to the electrode, followed by an impedance measurement and the impedances of the individual fields on the electrode were checked.

During this time, 10 mΐ of the virus suspension and 10 mΐ of the bacterial suspension were added to 50 mΐ of buffer obtained according to example 2. The solution was mixed and incubated at room temperature for 1 minute. Then 60 mΐ of the mixture prepared in such way was applied on the electrode by adding the solution to the measurement buffer obtained according to example 2

The impedance measurement has commenced. The results are shown in fig. 1. The presence of pathogens in the sample was confirmed by the PCR reference method (Fig. 2A and 2B).

Negative controls:

The study of sensor interaction on gold support with negative samples in the form of night culture of Haemophilus influenzae , Pseudomonas aeruginosa , Bordetella parapertussis and Streptococcus pneumoniae bacteria is carried out as follows:

150 mΐ of measurement buffer was applied to the electrode modified with HSCH ₂ (CH ₂ ) ₈ CH ₂ - C ONH-HTIHGAQ and HSCH ₂ (CH ₂ ) ₈ CH ₂ -CONH-MLPFRTD molecules, followed by calibration measurement.

After that the following bacteria solutions were applied to the electrode Haemophilus influenzae, Pseudomonas aeruginosa, Bordetella parapertussis and Streptococcus pneumoniae , from the night culture, OD ₆ oo=1.0.

The results are presented in fig. 3.

Example 10. Detection of S.pyogenes bacteria and influenza virus by a sensor containing electrodes modified with HSCH2(CH2) ₈ CH2-CONH-HTIHGAQ and HSCH2(CH ₂ )8CH2- CONH-MLPFRTD molecules in a 1 mM measurement buffer of potassium hexacyanoferrate (II)/(III) (K3[Fe(CN) ₆ ]/K [Fe(CN) ₆ ], 1:1) in PBS, 0.1 M pH 7 - reference example.

The measurement buffer was prepared according to example 8.

The electrode was prepared in the same way as in example 9. The positive test is a mixture of H1N1, (ATCC A/PR/8/34) virus suspended in the PBS buffer, titre 10 ⁷ CEID o/mL and S. pyogenes bacteria suspended in TBS, OD ₆ oo=TO.

The sensor was placed in the HDMI edge connector using a potentiostat containing a FRA card for impedance measurements (Autolab M204).

About 150 mΐ of measurement buffer obtained as in example 8 was applied to the electrode surface.

The first step of measurement has commenced - calibration of the sensor containing electrodes. 150 mΐ of measurement buffer was applied to the electrode, followed by impedance measurement and the impedances of individual fields on the electrode were checked.

During this time, 10 mΐ of the virus suspension and 10 mΐ of the bacterial suspension were added to 50 mΐ measurement buffer. The solution was mixed and incubated at room temperature for 1 minute. Then 60 mΐ of the mixture prepared in such way was applied to the electrode adding the solution to the measurement buffer. Impedance measurement began. The results are shown in fig. 4.

The obtained results show the influence on the electrode only on the channel modified with a molecule that recognizes S. pyogenes. There is no effect on the flu virus. This example excludes the possibility of using a reference buffer to test for both pathogens in single sample, e.g. in a swab from a patient with an upper respiratory tract infection.

Example 11. Measurement of interaction of antibody modified electrodes in buffer solution using the Haemophilus influenzae pathogen as an example

Steps of the electrode preparation for pathogen detection with the gold electrode using Haemophilus influenzae specific antibodies as an example

The gold electrodes were cleaned with 0.5 M H2SO4 for 30 minutes before the measurement. Modification of the gold surface, which is ultimately to be sensitive to the presence of protein D found in Haemophilus influenzae bacteria, consisted in covalent connection of anti-protein D antibodies using amide bond formed with a linker in the form of gluratinous aldehyde (GA) previously attached to the monolayer formed by 4-aminothiophenol (4-ATP) on gold (Au) support (Fig. 5.)

In the first step of modification Au electrodes were immersed in 0.1 M ethanol solution 4-ATP for 16 h at the temperature of 5-6°C. The phenomenon of creating a self-assembled monolayer (SAM) was applied, in which the thiol groups derivatives from 4-ATP were chemisorbed on the gold support forming the Au-S bond. The presence of the monolayer of free amine group - NFh on the surface allows for the connection of glutaraldehyde in the second step of modifications. For this purpose, 2.5% of aqueous GA solution into which modified Au/4-ATP electrodes were immersed and incubated for 15 min in a darkened place at room temperature. The linker formed in such way on the gold surface (Au/4-ATP/GA) was used in the third step of modifications to connect anti-protein D antibodies by forming an amide bond between - COOH groups derived from GA and the -NFh groups of antibodies. Incubation of the modified electrode Au/4-ATP/GA was performed in a solution of anti- protein D antibodies in PBS with a concentration of 21.5 pg/ml at 5-6°C for 1 h. To block the non-specific sensor response, the free spaces had to be blocked with neutral protein. In the last step 0.5% aqueous solution of bovine serum albumin (BSA) raw protein was used. The BSA solution was added dropwise on the modified Au/4-ATP/GA/anti- protein D electrode and incubated for 30 min at 5-6°C. The steps of sensor preparation using cyclic voltammetry CV and EIS are presented in fig. 6 - CV and fig. 7. EIS, respectively).

Testing the interaction of a ready-made sensor with a positive sample in the form of protein D present in Haemophilus influenzae bacteria

After steps of modification with antibody D and BSA, the sensor was measured for interaction with a positive sample (Fig. 8A.) in the form of protein D found in Haemophilus influenzae bacteria.

Testing the interaction of the ready-made sensor with a positive sample in the form of Haemophilus influenzae bacteria

After steps of modification with antibody D and BSA, the sensor was measured for interaction with a positive sample (Fig. 8B.) in Haemophilus influenzae bacteria.

Testing the interaction of the ready-made sensor with a negative sample

Subsequent to steps of modification with antibody D and BSA, the sensor was measured for interaction with negative samples in the form of bacteria that do not contain, on their surface, protein specific for anti-protein D antibodies (Fig. 9.):

A) Pseudomonas aeruginosa bacteria (P. aeruginosa )

B) Streptococcus pyogenes bacteria (S. pyogenes)

C) Streptococcus pneumoniae bacteria (S. pneumoniae)

Example 12: Testing for influenza virus and S. pyogenes bacteria in a swab taken from a patient with an upper respiratory tract infection

A system based on designed sensors can be used to detect pathogens in swabs from patients. In case of upper respiratory tract infection, a swab is taken from the throat.

The quality of the swab determines the quality of the obtained results, which is why swab control in the form of mucin protein detection was added in the sensor.

Eight-channel electrodes were modified according to the procedure described in Example 2. Molecules were used to detect Streptococcus pyogenes bacteria (FlSCF^CFysCFh-CONFl- SPAKPHSFYTGS), influenza virus (HSCH ₂ (CH2)8CH ₂ -CONH-FSTDYAWTAEAT) and mucin (HSCH ₂ (CH ₂ ) ₈ CH ₂ -CONH-TYNYDMPLRGRA).

The electrode was placed in the socket of an impedance spectrometer. Approximately 150 mΐ of measurement buffer (100 mM Tris 6.2 mM potassium hexacyanoferrate (II) (III), 0.1% vol. Tween 20) was applied on the electrode surface. The first step of the measurement commenced - calibration of the electrode. A swab stick with a collected flu-infected patient's swab (confirmed by PCR) was placed in 400 mΐ measurement buffer, incubated for 1 minute. After 1 minute of incubation the swab stick was removed from the buffer and 60 mΐ of solution was applied onto the electrode. Impedance measurement was performed on Autolab M204 impedance spectrometer. The results presented in fig. 10 indicate correct swab collection from the patient and the presence of both the flu virus and S. pyogenes bacteria. The presence of pathogens in the sample was confirmed by PCR reference method (Fig. 2A and 2B).

The developed buffer solution is used in electrochemical impedance spectroscopy for the detection of pathogenic bacteria and viruses, as demonstrated in the above examples.

These examples show that the developed buffer is universal to study both bacterial and viral biomarkers. The composition of the buffer allows for its use in electrochemical impedance spectroscopy to identify multiple pathogens simultaneously in a single sample.