Ultra-Fast Impedimetric Immunoassay for Detection of Streptococcus agalactiae Using Carbon Electrode with Nanodiamonds Film

This publication presents the results of work on the development of a quick and cheap electrochemical immunosensor for the diagnosis of infections with the pathogen Streptococcus agalactiae. The research was carried out on the basis of the modification of the well-known glassy carbon (GC) electrodes. The surface of the GC (glassy carbon) electrode was covered with a film made of nanodiamonds, which increased the number of sites for the attachment of anti-Streptococcus agalactiae antibodies. The GC surface was activated with EDC/NHS (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-Hydroxysuccinimide). Determination of electrode characteristics after each modification step, performed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS).


Introduction
Streptococcus agalactiae (also known as Group B Streptococcus or GBS) belongs to bacteria that are part of human microflora from the group of cocci. They inhabit the digestive system and the genitourinary tract. This streptococcus occurs in about 10-30% of healthy women and is usually a harmless bacterium that is naturally part of the female genital microbiota [1]. Streptococcus agalactiae is dangerous from the point of view of a woman of childbearing age and during pregnancy [2]. There are many changes in the reproductive organs of a pregnant woman, which significantly increase the risk of excessive growth of this bacterium. This is mainly due to the change in the pH vagina and hormonal changes (increase in estradiol) [3]. The infection may be mild and manifest with vaginal infections, or in severe cases, inflammation of the membranes, placenta or uterus may occur, which may result in premature birth, postpartum endometritis or even puerperal fever, which is a life-threatening condition [4]. Pregnant women colonized with GBS can transmit the bacteria to their newborns at the time of birth. The GBS colonization of newborns is caused by a vertical transmission through an oral cavity to a digestive and respiratory system and then by the bloodstream to distant organs. The risk of a newborn infection from the carrier mother is close to 70% [5]. Thus, GBS testing is especially important for pregnant women because these microorganisms may threaten the course of pregnancy and the newborn after its termination. S. agalactiae represents the main pathogen responsible for invasive infections and is the cause of sepsis and meningitis in newborns and young infants. In approximately 80% of cases, neonatal infection develops in the first week of life and is referred to as early-onset GBS disease. Late-onset GBS disease is defined as GBS infection in infants three months of age or older. The most common infections affecting these children are pneumonia, sepsis or meningitis [6,7]. Clinical and epidemiological Table 1. A comparison of the analytical characteristics of the immunosensors developed in this work with relevant immunosensors for S. agalactiae detection based on the literature.

Instrumentation
Potentiostat-galvanostat system (PalmSens4, Palmsens, Houten, The Netherlands), three-electrode assembly (Lambda System, Warsaw, Poland), glassy carbon disk electrode (3.0 mm in diameter, Mineral, Poland was used as a working electrode and Ag/AgCl/ 0.1 M KCl was used as a reference electrode, while Pt mesh served as an auxiliary electrode.

Preparation of ND-PS Dispersion and Sensor Fabrication
Potato starch dispersion was prepared in a proportion of 1.0 g of powdered potato starch to 100 mL of 5% acetic acid solution. The mixture was left under stirring at a temperature of 85 • C for 2 h until complete homogenization and a whitish transparent liquid was obtained. The resulting dispersion was stored under refrigeration. Then, 1.0 mg of NDs was added in 1.0 mL of MS, which remained in constant magnetic stirring for 2 h until complete dispersion homogenization. GCE was carefully polished with activated alumina, 1:1 proportion (v/v), for 5 min on a piece of clean cotton fabric and rinsed thoroughly with ultrapure water. Then, 5.0 µL of that dispersion was dropped on the GCE surface, and the solvent was evaporated at room temperature for 2 h.

Immunosensor Fabrication
Prior to electrochemical measurements, glassy carbon electrodes with nanodiamonds film were cleaned using ethanol and demineralized water. Then, the electrodes were modified by polarizing the sample eleven times in a previously prepared deoxidized solution of diazonium salt [43]. 20 mg of 4-ABA was dissolved in 2 mL of 37% HCl (stirring for 15 min-average stirring speed 400 rpm). Then, it was cooled to 0 • C. Next, 2 mL of demineralized water was added to the mixture. The mixture was then stirred for a further 15 min to dissolve the precipitated 4-ABA chloride. Then 25 mg NaNO 2 dissolved in 3 mL ddH2O was added dropwise for 30 min. After the addition of sodium nitrite, the compounds were stirred at 0 • C for about 10 min.
Modification of the GC surface was achieved by voltammetric electroreduction of the aryldiazonium reagents. The nitrosonium ion is formed in formation, which subsequently activates the amino group on 4-ABA. During CV sweeping, irreversible reduction peaks occur at a potential around 0.2 V. These peaks form due to the reduction of the diazonium precursor reagents by single electron transfer. Modification by electrode polarization from 0 V vs. Ag/AgCl to −1 V vs. Ag/AgCl five times at a speed of 100 mV/s was prepared with deoxidized diazonium salt solution with an Ag/AgCl (3M KCl) electrode as reference electrode (RE), and a platinum mesh as a counter electrode (CE).
The samples were then washed with a strong stream of ddH2O and dried with a stream of argon, and 50 mM EDC and 100 mM NHS was placed on electrodes. This process lasted an hour and occurred at 4 • C. The samples were then washed with ddH2O, incubated with 10 µL of 0.1 µg/mL antibody solution and left for 24 h at 4 • C.

Electrochemical Measurements
The cyclic voltammetry and electrochemical impedance spectroscopy (EIS) were conducted using a Palmsens 4 potentiostat/galvanostat system (Methrom, Autolab, Utrecht, The Netherlands) in the standard three-electrode configuration. Glassy carbon electrode (Mineral, Poland) was used as a working electrode (GCE ∅ 3 mm), modified with a film from the dispersion of NDs in PS acid solution, Ag|AgCl (3.0 mol L −1 KCl) as a reference electrode; and wire of platinum as a counter electrode (Pt).
All the electrochemical tests were carried out in 5 mM K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ] in 0.01 M PBS that was previously deaerated. In case of the electrochemical impedance spectroscopy measurements (EIS), the frequency ranged from 10 kHz to 1 Hz with 40 points. The amplitude of the AC signal was 10 mV. Each potential was held constant for 60 s before each measurement to obtain steady-state conditions. Obtained data were subjected to the analysis using EIS Spectrum Analyzer according to the proposed electric equivalent circuit (EEQC).

Electrochemical Characterization of Immunosensor
The electroactive area was estimated for GCE and GCE with nanodiamonds film, from I p = 2.69 × 10 5 AD 1/2 n 3/2 ν 1/2 C where I p is the anodic or cathodic peak current, n is the number of electrons transferred (n = 1), A is the electroactive surface area, D is the diffusion coefficient (D = 7.6 × 10 −6 cm 2 s −1 for redox probe in 0.01 M PBS solution and C is the redox concentration. Figure 2 shows a comparison of cyclic voltammograms between the GC and GC with NDs electrodes in the presence of 5 mM K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ] in 0.01 M PBS solution at a scan rate of 10 mV/s-500 mV/s. The difference of peak potentials (∆Ep) for the GC with NDs electrode was 200 mV, while for the GCE, it was 243 mV, showing an improvement in reversibility for the redox pair for the GC electrode with nanodiamonds film. These results indicate that the GC with NDs exhibits remarkably better electrochemical performance than the GC. These results indicate that the GC with NDs exhibits remarkably better electrochemical performance than the GC. Checking the correctness of successive modifications of the electrode was carried out by CV and EIS. All electrochemical measurements made were using a solution buffer in PBS solution, pH 7.4, containing 5 mM K3[Fe(CN)6] and 5 mM K4[Fe(CN)6]. The applied redox system enables the analysis of changes in the kinetics of electron transfer on the surface, thanks to which we are able to notice changes after each modification stage, which allows us to determine the correctness of each step. CV measurements were performed in the potential ranges from −0.20 V to 0.60 V with a scan rate of 0.1 V/s. The obtained potential differences are related to the electrochemical window characteristic for each electrode. The obtained CV spectra provide information about changes in charge transfer, and EIS measurements inform about changes in resistance that occur on the electrode surface. Checking the correctness of successive modifications of the electrode was carried out by CV and EIS. All electrochemical measurements made were using a solution buffer in PBS solution, pH 7.4, containing 5 mM K 3 [Fe(CN) 6 ] and 5 mM K 4 [Fe(CN) 6 ]. The applied redox system enables the analysis of changes in the kinetics of electron transfer on the surface, thanks to which we are able to notice changes after each modification stage, which allows us to determine the correctness of each step. CV measurements were performed in the potential ranges from −0.20 V to 0.60 V with a scan rate of 0.1 V/s. The obtained potential differences are related to the electrochemical window characteristic for each electrode. The obtained CV spectra provide information about changes in charge transfer, and EIS measurements inform about changes in resistance that occur on the electrode surface.
Data from the electrochemical measurements during carbon surface with Nanodiamonds modification are shown in Figure 3A. In Figure 3B, it is easy to follow the changes recorded during the successive steps of the sensor modification recorded by the CV. In addition to the previously discussed changes after incubation in Nanodiamonds, there is a clear decrease in the height of the current peaks from the oxidation and reduction in [Fe(CN) 6 ] 3−/4− by 16 µA. Data from the electrochemical measurements during carbon surface with Nanodia monds modification are shown in Figure 3A. In Figure 3B, it is easy to follow the change recorded during the successive steps of the sensor modification recorded by the CV. I addition to the previously discussed changes after incubation in Nanodiamonds, there i a clear decrease in the height of the current peaks from the oxidation and reduction i [Fe(CN)6] 3−/4− by 16 µA. However, when it is hard to see the binding receptor to the surface and saturatin the free sites with BSA, it is much easier to interpret changes occurring on the sample from the impedance spectra shown in Figure 3B. To better present the data, the impedanc spectra are shown in the range from 10 kHz to 1 Hz. To improve data analysis from th EIS, they were fitted to an equivalent electrical circuit (EEC), which is shown in Figure 3C and the results are shown in Figure 3C with the chi-square parameter interpreted as goodness of fit.

Detection of Streptococcus Agalactiae Protein
In order to obtain a satisfactory level of resistance changes on the tested electrode the influence of the incubation time of the analyzed sample on the level of the obtaine response was checked. For this purpose, analyzes were carried out in which the incubatio time of the analyzed samples was as follows: 3 min, 5 min, 7 min and 10 min (Figure 4). However, when it is hard to see the binding receptor to the surface and saturating the free sites with BSA, it is much easier to interpret changes occurring on the sample from the impedance spectra shown in Figure 3B. To better present the data, the impedance spectra are shown in the range from 10 kHz to 1 Hz. To improve data analysis from the EIS, they were fitted to an equivalent electrical circuit (EEC), which is shown in Figure 3C, and the results are shown in Figure 3C with the chi-square parameter interpreted as a goodness of fit.

Detection of Streptococcus Agalactiae Protein
In order to obtain a satisfactory level of resistance changes on the tested electrodes, the influence of the incubation time of the analyzed sample on the level of the obtained response was checked. For this purpose, analyzes were carried out in which the incubation time of the analyzed samples was as follows: 3 min, 5 min, 7 min and 10 min (Figure 4). recorded during the successive steps of the sensor modification recorded by the CV. addition to the previously discussed changes after incubation in Nanodiamonds, there a clear decrease in the height of the current peaks from the oxidation and reduction [Fe(CN)6] 3−/4− by 16 µA. However, when it is hard to see the binding receptor to the surface and saturati the free sites with BSA, it is much easier to interpret changes occurring on the sample fro the impedance spectra shown in Figure 3B. To better present the data, the impedan spectra are shown in the range from 10 kHz to 1 Hz. To improve data analysis from t EIS, they were fitted to an equivalent electrical circuit (EEC), which is shown in Figure 3 and the results are shown in Figure 3C with the chi-square parameter interpreted as goodness of fit.

Detection of Streptococcus Agalactiae Protein
In order to obtain a satisfactory level of resistance changes on the tested electrod the influence of the incubation time of the analyzed sample on the level of the obtain response was checked. For this purpose, analyzes were carried out in which the incubati time of the analyzed samples was as follows: 3 min, 5 min, 7 min and 10 min (Figure 4) The results obtained for the following times are presented in Table 3 below. The analysis of the obtained results indicated no significant differences in the level of received responses to the sample. However, it can be argued that the optimal range of incubation will be up to 5 min due to the fact that after this time, the level of response to the sample did not change or was lower. For this reason, the final incubation time for each of the positive and negative control samples analyzed was 5 min.

Biosensor Selectivity, Repeatability and Stability Studies
Mycoplasma hominis, Ureaplasma, Streptococcus agalactiae, Gardnerella vaginalis bacteria were used as potentially interfering bacteria to investigate the selectivity of the presented immunosensor. The bacteria concentration was kept in the same order of magnitude to receive comparable results. After 5 min incubation, the EIS spectra were recorded. According to Figure 5, all negative controls did not give a substantial impedance increase, the percentage change of Rct did8 not exceed 25% for both single samples, and this value was established as a threshold for the distinction between positive and negative samples. The results obtained for the following times are presented in Table 3 below. The analysis of the obtained results indicated no significant differences in the received responses to the sample. However, it can be argued that the optimal r incubation will be up to 5 min due to the fact that after this time, the level of resp the sample did not change or was lower. For this reason, the final incubation time of the positive and negative control samples analyzed was 5 min.

Biosensor Selectivity, Repeatability and Stability Studies
Mycoplasma hominis, Ureaplasma, Streptococcus agalactiae, Gardnerella v bacteria were used as potentially interfering bacteria to investigate the selectivit presented immunosensor. The bacteria concentration was kept in the same order nitude to receive comparable results. After 5 min incubation, the EIS spectra w orded. According to Figure 5, all negative controls did not give a substantial im increase, the percentage change of Rct did8 not exceed 25% for both single samp this value was established as a threshold for the distinction between positive and n samples. After a glassy carbon electrode with nanodiamonds film modification, it wa in an electrochemical cell, and EIS spectra were recorded until system stabilizat observed. The stability of the sensor was verified by two additions of PBS to excl specific interactions. In the case of the immunosensor, all pathogens sample diss 0.01 M PBS was firstly incubated on the electrode surface for a given time, rinsed w and immediately immersed in fresh 5 mM K3[Fe(CN)6]/K4[Fe(CN)6]/0.01 M PBS for EIS measurement. Figure 5 shows the impedance spectrum recorded during quent additions of the protein solution with increasing concentrations, wh After a glassy carbon electrode with nanodiamonds film modification, it was placed in an electrochemical cell, and EIS spectra were recorded until system stabilization was observed. The stability of the sensor was verified by two additions of PBS to exclude unspecific interactions. In the case of the immunosensor, all pathogens sample dissolved in 0.01 M PBS was firstly incubated on the electrode surface for a given time, rinsed with PBS and immediately immersed in fresh 5 mM K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]/0.01 M PBS solution for EIS measurement. Figure 5 shows the impedance spectrum recorded during subsequent additions of the protein solution with increasing concentrations, which was preceded by two-times additions of its solvent (PBS) as a negative control Table 3 presents GC-based immunosensor response after incubation in the protein sample, expressed as charge transfer resistance change (∆R ct ). All R ct change values were calculated from the equation: where Rct Test is for the sample and Rct Basic is for the fully prepared immunosensor. The EIS was used to investigate the metrological performance of the biosensor detecting the SARS-CoV-2 virus protein N by spotting the solutions with different concentrations (1.38 pg/mL, 13.8 pg/mL, 13.8 ng/mL, 0.138 ng/mL, 1.38 µg/mL) on the surface of electrodes and incubating them for optimal time ( Figure 6). preceded by two-times additions of its solvent (PBS) as a negative control Table 3 presents GC-based immunosensor response after incubation in the protein sample, expressed as charge transfer resistance change (ΔRct). All Rct change values were calculated from the equation: where Rct Test is for the sample and Rct Basic is for the fully prepared immunosensor. The EIS was used to investigate the metrological performance of the biosensor detecting the SARS-CoV-2 virus protein N by spotting the solutions with different concentrations (1.38 pg/mL, 13.8 pg/mL, 13.8 ng/mL, 0.138 ng/mL, 1.38 µg/mL) on the surface of electrodes and incubating them for optimal time ( Figure 6).
The limit of detection was calculated from the relation LOD = 3 × SD/slope, where SD is the standard deviation in the low concentration range. For all tested surfaces, we obtained a wide linear range of concentrations from 1.38 ug/mL to 1.38 pg/mL.

Conclusions
This work presented the design and characterization of Streptococcus agalactiae antibodies immobilization onto a glassy carbon electrode with nanodiamonds film for impedimetric detection of Streptococcus agalactiae protein. The assay could detect Streptococcus agalactiae protein at concentrations as low as 1.38 pg/mL with a linear detection range of 2.92 ng/mL (R 2 = 0.98). The advantage of this 'one-step' diagnostic assay relative to an ELISA or mass spectrometry is a rapid and sensitive measurement of antigen binding to nanodiamonds film. This provides a proof of concept that we intend to use to develop a clinical test with swabs from gynecological patients in order to determine whether Streptococcus agalactiae bacteria detection in patient fluid provides any prognostic indicator. Therefore, the electrode modified with NDs has shown very promising results in the electrochemical sensing of Streptococcus agalactiae. The limit of detection was calculated from the relation LOD = 3 × SD/slope, where SD is the standard deviation in the low concentration range. For all tested surfaces, we obtained a wide linear range of concentrations from 1.38 ug/mL to 1.38 pg/mL.

Conclusions
This work presented the design and characterization of Streptococcus agalactiae antibodies immobilization onto a glassy carbon electrode with nanodiamonds film for impedimetric detection of Streptococcus agalactiae protein. The assay could detect Streptococcus agalactiae protein at concentrations as low as 1.38 pg/mL with a linear detection range of 2.92 ng/mL (R 2 = 0.98). The advantage of this 'one-step' diagnostic assay relative to an ELISA or mass spectrometry is a rapid and sensitive measurement of antigen binding to nanodiamonds film. This provides a proof of concept that we intend to use to develop a clinical test with swabs from gynecological patients in order to determine whether Streptococcus agalactiae bacteria detection in patient fluid provides any prognostic indicator. Therefore, the electrode modified with NDs has shown very promising results in the electrochemical sensing of Streptococcus agalactiae.