Advancing palladium PEDOT molecularly imprinted nanoelectrochemical sensing interface for selective dual detection of Helicobacter pylori BabA antigen and antibody

Abstract

Helicobacter pylori (H. pylori) is a gram-negative microaerophilic bacterium that secretes several endotoxins to induce stomach and duodenum diseases. Herein, a molecularly imprinted polymer (MIP) based electrochemical and immuno-sensor was developed for the determination of H. pylori-specific BabA antigen (^BabAAg) and antibody (^BabAAb), respectively. The foremost step is the electropolymerization of palladium nanoparticles (PdNPs) and poly (3,4-ethylenedioxythiophene) (PEDOT) onto the electrode surface. The second step involves the fabrication of an electropolymerization-based ^BabAAg recognition platform (MIP) at the screen-printed gold electrode surface. Thirdly, anti-^BabAAb was used to further cross-validate MIP_BabA and ^BabAAg binding on the fabricated electrode. The fabricated MIP_BabA/PdNPs/PEDOT sensor detects the variable concentrations of ^BabAAg under electrochemical studies. The imprinted polymer was characterized with scanning electron microscopy, Brunauer–Emmett–Teller analysis, and X-ray photoelectron spectroscopy techniques. The electrochemical studies demonstrated the high sensitivity of the sensor with a low detection limit of 0.1 ng/ml in a linear dynamic range of 0.1–50 ng/ml BabA. The prospect of work includes the integration of a developed detection technique with a lab-on-a-chip kit, as accurate and early detection of H. pylori-associated biomarkers such as BabA is important for timely diagnosis and monitoring of gastric disorders, including gastric cancer.

Supplementary Information

The online version contains supplementary material available at 10.1186/s11671-026-04720-5.

Introduction

Electrochemical sensors have gained significant attention due to their high sensitivity, rapid response, and potential for point-of-care applications [1]. Recently, several enzymes, antigens-antibodies, DNA, and microorganism-based electrochemical sensors have been developed, but the implementation of all these techniques in a clinical setup remains limited [2]. However, a clinically based sensor needs to be stable at various pH, temperature, and environmental conditions, reliable immobilization methods, short response time, and an extended linear range without any false-positive results [3]. Therefore, developing new platforms to overcome all these limitations is a primary target for research teams all around the world.

In order to overcome these problems, molecular imprinting polymer (MIP) technology, which is a template-based technique, has been used. It is gaining a lot of attention due to its higher selectivity and stability in analytical chemistry [4, 5]. MIP can replace the biorecognition element in biosensors. The principle behind MIP-based sensors is to create an analyte mimicking molecular lock from a polymer that has binding sites complementary to the target analyte in terms of shape, size, and functional groups [6]. The three-dimensional detection approach for analyte binding to a polymer matrix-based recognition site can be designed with the initial interaction of a functional monomer with a target analyte, before applying a crosslinker [7]. The MIP has unique characteristics like structure predictability, recognition specificity, and application universality. Recently, many kinds of sensors, whether chemical or biological, have been introduced that are based on MIPs. Compared to antibodies, enzymes, and hormone receptors, MIP demonstrates higher stability and lower cost while having a similar binding affinity with the target of a polyclonal antibody [8]. Several studies have been conducted to develop MIP for small molecules, but MIP development for macromolecules, such as proteins, has been difficult due to the large molecular size, inactivation, complexity, and solubility of the proteins [9]. To date, several MIP-based electrochemical sensors have been developed to determine the presence of microorganisms in the healthcare system. [10, 11].

H. pylori is a microaerophilic gram-negative bacterium that is mainly responsible for several gastric diseases like gastritis, duodenal ulcers, gastric and mucosa-related lymphoid tissue (MALT) lymphoma, non-Hodgkin lymphoma, and gastric cancer [12]. H. pylori secretes several types of toxins in gastric cells to establish the infection, for example, cytotoxin-associated gene A (CagA), vacuolating cytotoxin gene A (VacA), and several outer membrane proteins. The blood group antigen-binding adhesion (BabA) is one of the outer membrane chimeric proteins that is mainly responsible for cell adhesion to host cells and further facilitates bacterial colonization [13]. These toxins are exploited as biomarkers for the detection of H. pylori. Although, there are some conventional methods present which are utilized for the detection of bacteria such as histology, rapid urease test (RUT), microbiological culture, polymerase chain reaction (PCR), endoscopy, stool antigen test, serology and urea breath test (UBT) but these techniques are expensive, time-consuming and require expertise to analyze the results [14]. Therefore, to monitor and manage H. pylori-related diseases or infections, a few studies have been conducted to develop H. pylori-specific sensors [15, 16], though they have some limitations, e.g., high cost and instability. Therefore, we have designed a new approach for the detection of H. pylori-specific BabA antigen (^BabAAg) and antibody (^BabAAb) through electrochemical immunosensing on nanomaterial-coated MIP-based gold screen printed electrode (Au-SPE). In this study, we have proposed to cross-check the analyte detection using target-specific antibodies.

The first and foremost step is the electropolymerization and electrodeposition of poly(3,4-ethylenedioxythiophene (PEDOT) and palladium nanoparticles (PdNPs) onto the Au-SPE. PdNPs and PEDOT are known for their large surface area, stability, and high electrical conductivity; thus, they are widely used. Afterward, the MIP was prepared with electrochemical polymerization of the appropriate monomer and cross-linker in the presence of ^BabAantigen as a template (MIP_template) over the PdNPs/PEDOT/Au-SPE. The elution of the template causes the formation of molecular sieves which mimic the ^BabAAg in terms of size and shape (MIP_BabA). The developed sensing platform consists of a polymer matrix that can be bound with ^BabAAg (^BabAAg@MIP_BabA) in analytical samples, and further addition of ^BabAAb in solution was allowed to the ^BabAAg and Ab interaction (^BabAAb-^BabAAg@MIP_BabA). Herein, we have performed the study with BabA-specific Ag and Ab. Thus, this single platform is suitable for both Ab and Ag molecules. There are two ways to detect H. pylori-specific BabA protein on a MIP sensing platform: (1) electrochemical studies were performed for direct recognition of ^BabAAg on MIP_BabA/PdNPs/PEDOT/Au-SPE platform, and further, (2) the same platform (^BabAAg@MIP_BabA/PdNPs/PEDOT/Au-SPE) as mentioned in point 1, can be exposed to anti-^BabA Ab after exposure to ^BabAAg on the platform.

Due to intrinsic properties like the binding process of antigen–antibody interaction, by directly monitoring, the electrochemical immunosensor has drawn broad consideration. Therefore, the necessities of the nanocomposite used in the sensor should be very stringent. Besides this, it should be a good carrier altogether and should not obstruct the electron exchange, to append proteins; it should have active sites, and a vast surface area to facilitate the immobilization of more antibodies onto the desired nanomaterials [17].

Materials and methods

Chemicals & instruments

BabA antigen and antibodies were obtained from the Max von Pettenkofer Institute of Hygiene and Medical Microbiology, Ludwig Maximilian University, Munich, Germany. The chemicals 3,4-ethylenedioxythiophene (EDOT), K₂PdCl₄, ethylene glycol, poly(N-vinyl-2-pyrrolidone) (PVP), and BSA were sourced from Sigma Aldrich, USA. The monomer methacrylic acid (MAA, 99%), the crosslinker ethylene glycol dimethacrylate (EGDMA, 97%), the initiator azobisisobutyronitrile (AIBN), and the solvents acetonitrile (ACN, 99.5%) and methanol were purchased from Sigma Aldrich, India, and used without further purification. Blood samples were collected from Bio-diagnostics Lab, Rohini, New Delhi, India. Additional chemicals, such as K₃[Fe(CN)₆], K₄[Fe(CN)₆], and KCl, were procured from Sisco Research Laboratory (SRL), Mumbai, India. All remaining reagents were of analytical grade, and all aqueous solutions were prepared using double-distilled water.

Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were conducted on a Bio-Logic Science Instruments SP-200 system equipped with EC-Lab® software and its impedance analysis module. A screen-printed gold electrode (Au-SPE) obtained from PalmSens was used as the solid support for the experiments. Each electrode, fabricated via screen printing, incorporated a gold counter electrode, a silver pseudo-reference electrode, and a circular gold working electrode. The geometric area of the working electrode used in this study was 7.07 mm², corresponding to a 3 mm diameter electrode.

Cell culture and purification of H. pylori BabA protein

We have procured the purified form of BabA antigen and aliquots of BabA antibodies from the laboratory.

The cell culture maintenance and purification of the H. pylori strain were performed as previously reported [18]. The purified form of H. pylori was stored at − 20 °C.

Electro-polymerization of PEDOT over Au-SPE

A three-electrode cell configuration was used for the electrochemical experiments. Electro-polymerization of EDOT on the Au-SPE was performed in a deaerated aqueous EDOT solution (0.1% w/v) containing NaPSS (0.7% w/v). To fully dissolve the designated amount of EDOT, the solubility of the deaerated aqueous medium had to remain below the solubility limit of EDOT in water (15 mM at 25 °C). Cyclic voltammetry was then conducted at a scan rate of 20 mV/s for 10 cycles within the potential range of –0.9 to 1.2 V [19]. A slow scan rate was intentionally selected to achieve uniform polymer growth on the electrode surface. The scan rate also influences the thickness of the diffusion layer formed above the electrode. Typically, slower scan rates allow the diffusion layer to expand and become thicker, while faster scan rates generate sharp peaks with higher currents and elevated resistances, which can introduce distortions [20].

Electrodeposition of PdNPs/PEDOT on Au-SPE

PdNPs were electrochemically deposited by immersing the PEDOT-modified Au-SPE into a solution containing 1 × 10⁻³ M K₂PdCl₄ and 0.5 M H₂SO₄. The electrode was subjected to 10 cyclic voltammetric scans between − 0.25 and 1.2 V at a scan rate of 50 mV/s.

Synthesis of molecularly imprinted polymer for BabA template

The MIP was synthesized by bulk polymerization for morphological and compositional characterization [21]. Electrochemical polymerization of MIP_template on Au-SPE was done by the CV technique. Briefly, the optimized ratio of MAA monomer, EGDMA crosslinker, and 1:1000 dilution of the target analyte, ^BabAAg was mixed in ACN solvent and stirred at 50 °C for 12–16 h for bulk polymerization. Similarly, the same experimental conditions were applied to prepare a non-imprinted polymer (NIP) except for using ^BabAAg in the polymeric solution. The end product was crushed, and additional moisture was removed after drying in a vacuum overnight. Further, target analyte elution was performed by dispersing MIP in a 4:1 ratio of methanol and acetic acid at first, and later pure methanol was used for MIP washing. The end product (MIP_BabA) was kept in inert conditions till further use. The morphological characterization of NIP, MIP before (MIP_template) and after ^BabAAg elution (MIP_BabA) was performed by SEM, XPS, and BET analysis of the polymer matrix. Furthermore, the different steps involved in the fabrication of the BabA-MIP-based sensing platform are represented in Scheme 1. The illustration demonstrates the stepwise modification of Au-SPE in order to develop an MIP-based sensing platform. The nanomaterials-coated working electrode modified with BabA antigen-specific MIP, used for dual confirmation of H. pylori based on electrochemical detection.

Scheme 1.

Schematic illustration of the steps involved in the fabrication of the BabA MIP-based sensing platform. PEDOT and Pd nanoparticles are electrodeposited onto Au-SPE, followed by electropolymerization of the MIP layer in the presence of BabA template. After template removal, specific binding cavities are formed for BabA-Ag recognition. The platform enables dual electrochemical detection via antigen rebinding and subsequent antibody interaction

Electropolymerization of MIP_template over PdNPs/PEDOT/Au-SPE

The MIP_template was electropolymerized over PdNPs/PEDOT/Au-SPE using a solution containing ^BabAAg as template, monomer, and crosslinker. A three-electrode system, consisting of a working electrode, reference electrode, and counter electrode, was used. Electropolymerization was performed using CV with a scan rate of 50 mV/s, scanning from − 0.2 to 0.6 V for 15 polymerization cycles. The peak cathodic current is decreased with every cycle. The reason behind this could be the initiation of acrylamide monomer’s electro-polymerization by the synthesis of free radicals and deposition of polyacrylamide subsequently on the electrode surface. To wash out the template from MIPs, we optimized the conditions, including cycling the voltage post-polymerization. The fabricated platform was dried for a few hours for strong binding of the polymer matrix with Au-SPE. To elute the ^BabAAg from the fabricated MIP_template/PdNPs/PEDOT/Au-SPE platform, an electrode potential from − 0.4 to 0.4 V was applied for 15 cycles in the presence of a 4:1 ratio mixture of methanol and acetic acid. Similar electrochemical conditions were applied in the second step of washing in the presence of methanol. For washing, a voltage change is essential to remove the template. Overall, 15 cycles were used to remove the template from MIP without disturbing the integrity of electro-polymerized MIPs, apparently. Furthermore, at the time of screening/detection, we have to use a different range of voltage so that we can find the optimum current response due to the synergistic effect of the various materials deposited on the electrode surface. At last, the electrode was kept for drying for a few hours for further electrochemical experiments. In this way, we have fabricated a ^BabAAg mimicking sensing platform, i.e., MIP_BabA/PdNPs/PEDOT/Au-SPE, which was used to study the response of the biosensor in the presence of ^BabAAg and ^BabAAb both.

Electrochemical response study using MIP_BabA/PdNPs/PEDOT/Au-SPE

CV and EIS studies were performed in 5 mM [Fe(CN)₆]^3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.0) at a scan rate of 50 mV/s for the characterization of the fabricated electrode. The electrochemical behavior of Au-SPE, deposited nanomaterials, NIP, MIP_template/PdNPs/PEDOT/Au-SPE, and MIP_BabA/PdNPs/PEDOT/Au-SPE (before and after removal of ^BabAAg) was studied by CV in the range of − 0.4 and + 0.4 V with a scan rate of 50 mV/s. The EIS experiments were performed and are represented by a complex Nyquist plot. EIS is an effective method for analyzing the interfacial characteristics of bio-recognition events, i.e., binding of analytes within the polymer matrix of MIP at the electrode surface. EIS is basically used to measure the impedance in a circuit in the form of a resistance unit, i.e., Ohms. In order to determine the effect of ^BabAAbs on MIP_BabA/PdNPs/PEDOT/Au-SPE, the 1:1 ratio of ^BabAAg and Abs was incubated with the fabricated electrode, and electrochemical changes were recorded.

Analytical performance of the fabricated electrode

To further evaluate the optimum conditions for the fabricated electrode, ^BabAAg was allowed to interact with MIP_BabA/PdNPs/PEDOT/Au-SPE, and similarly ^BabAAb with ^BabAAg@MIP_BabA/PdNPs/PEDOT/Au-SPE with concentration ranging from 0.1 to 50 ng/ml in 5 mM [Fe(CN)₆]^3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.0) at scan rate 50 mV/s. The 0.1 ng/ml ^BabAAg was also incubated with MIP_BabA/PdNPs/PEDOT/Au-SPE in every step of the concentration study. The performance of the fabricated electrode was analyzed with various pH, temperature, and scan rate via CV in the range of − 0.4 and + 0.6 V with a scan rate of 50 mV/s in 5 mM [Fe(CN)₆]^3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.0). Selectivity of the platform was assessed by comparing the developed BabA immunosensor's response to possible interfering biomolecules that might exist in biological matrices. Additionally, actual serum samples were examined and contrasted with the conventional ELISA method in order to evaluate the immunosensor's practical applicability. To confirm the accuracy of the developed platform, diluted human serum samples were spiked with known concentrations of BabA, and recovery tests were carried out.

Results and discussion

Characterization of synthesized BabA-MIP and NIP

Morphology study through SEM

The SEM images of NIP, MIP_template, and MIP_BabA shown in Fig. 1 denote the surface morphology of the prepared polymer matrix. The looser and uniform morphology of NIP (Fig. 1A) surface indicates the absence of ^BabAAg matching sites in the polymeric matrix, as there is no defined structure and pattern. In the case of MIP_template (Fig. 1B), the size of particles is relatively small in comparison to NIP. After the removal of ^BabAAg from the polymeric system, a surface with analyte mimetic pores on the MIP surface was observed (Fig. 1C) that is absent in MIP_template (Fig. 1B). This indicated prominent imprinting in the polymeric matrix, which allows interaction with the target ^BabAAg [22, 23].

Fig. 1.

SEM images illustrating the surface morphology of A non-imprinted polymer (NIP), B MIP_template, and C MIP_BabA after template removal. The NIP exhibits a relatively smooth and featureless surface, indicating the absence of specific binding sites. The MIP_template shows a denser morphology due to the presence of embedded BabA template. After template removal, MIP_BabA reveals distinct porous cavities (highlighted), confirming successful imprinting and formation of BabA-specific recognition sites

AFM analysis

The surface morphology and roughness parameters of the electrodes modified by nanomaterials and molecularly imprinted polymers (MIPs) were examined using Atomic Force Microscopy (AFM). Figure 2 displays the corresponding AFM images. The morphology of the nanomaterial-modified surface (Fig. 2A) was comparatively compact and uniform, with an average roughness (Ra) of 6.37 nm and a root-mean-square roughness (RMS) of 8.31 nm. The deposition of nanostructures, which increases the available surface area, is responsible for this smoother topology. The MIP-modified surface (Fig. 2B), on the other hand, showed a noticeably rougher texture with a Ra of 11.93 nm and a RMS of 14.76 nm. The polymerization of MIP is responsible for the increase in surface roughness. In addition to improving the film's effective thickness and topographic characteristics, these imprinted cavities on MIP act as specific binding sites for the target analyte. The successful imprinting process is amply supported by the notable difference in roughness values between the MIP layer and the nanomaterial layer.

Fig. 2.

Atomic force microscopy, A Nanomaterial deposition, B Nanomaterial and MIP deposition

BET analysis

The nitrogen adsorption–desorption analysis was performed to calculate the morphological changes in the sequential fabrication. The formation of the BabA binding site perhaps created a large number of microcavities and changed the pore structure of the polymer matrix. Figure 3 shows N₂ adsorption–desorption isotherms and pore sizes of NIP and MIP after ^BabAAg elution. The isotherms displayed in Fig. 3A indicated the IV-type curve with type-H3 hysteresis loop, which explains the hierarchical pore structure associated with meso and macropores morphology of NIP and MIP after ^BabAAg removal. On the other hand, Fig. 3B reveals that nitrogen adsorption–desorption isotherm that shows monolayer adsorption. It was also observed in BET analysis that the specific surface area of NIP (31.602 m²/g) is less than ^BabAAg eluted MIP (79.530 m²/g), and it is supported to use MIP in sensing applications despite their bulk counterpart [24]. Porosity is the fraction of pore volume to the whole volume. The pore size distribution curve in Fig. 3A shows a higher peak in the case of MIP_BabA. The insight into porosity of the polymeric system signifies mesoporous structure (2–7%) and the existence of nanocavities in the polymeric matrix [25].

Fig. 3.

Characterization of synthesized polymers using the BET analysis, A Pore size (red-NIP and black-BabA-Ag@MIP); B Volume (red-NIP and blue-BabA-Ag@MIP)

XPS analysis

XPS high-resolution spectra were recorded for deep analysis of surface chemical species present in different states of MIP synthesis. High-resolution XPS spectra for C1s and O1s are shown in Fig. 4 for MIP before and after template removal. The graphs were fitted with three peaks with the help of XPSPeak41 software. In C1s XPS spectra, the first peak was allotted for the C–C bonds aliphatic chain of the methacrylic acid monomer used to synthesize the MIP polymer matrix. The second and third peaks were assigned for C–O and C=O, respectively, present in the functional group of the monomer. Further, O1s spectra also showed three peaks for C–O, C=O, and a third for adsorbed water. Figure 4 shows C1s XPS spectra for MIP before and after template removal of the BabA template. It was clearly observed that there is no significant difference between the two states due to similarity in chemical environment at the core level of C1s, even after the removal of the template. The similarity between the atomic concentration of C–O and C=O for MIP before and after removal of template indicates that the chemical environment having carbon and oxygen in the polymer matrix was not affected by the absence of template ^BabAAg. Whereas the difference in peak intensities and atomic concentration of C–C bonds shows the presence of sp² and sp³ carbon atoms of ^BabAAg. The intermolecular interaction formed within MIP between the template and active cavities is a very weak interaction, so they do not contribute to the increase in binding energy. Whereas O1s spectra didn’t show any significant difference in the presence or absence of the template molecule [26, 27]. The binding energy and atomic percentage values of the chemical species are shown in Table 1. After washing, only minor shifts in binding energies and small reductions in atomic concentration were observed. These subtle changes confirm the removal of loosely bound template molecules and impurities, while the overall chemical composition of the MIP surface remained stable.

Fig. 4.

Characterization of synthesized polymers using high-resolution XPS spectra of C1s and O1s core levels for MIP before and after removal of template

Table 1.

The binding energy and atomic percentage values of the chemical species

C1s					O1s
MIP before washing			MIP After washing		MIP before washing			MIP After washing
Bond	Peak BE	Atomic concentration	Peak BE	Atomic concentration	Bond	Peak BE	Atomic concentration	Peak BE	Atomic concentration
C–C	281.8	69.86	281.92	67.31	O–C	531	39.581	531	39.581
C–O	283.3	16.786	283.28	16.786	O=C	529	26.17	529	25.72
C=O	285.9	0.314	285.81	0.314	Ads. H2O	527	0.733	527	0.733

Electrochemical deposition of MIP_template/PdNPs/PEDOT onto Au-SPE

Figure S1 depicts the electro-deposition of MIP_template/PdNPs/PEDOT over Au-SPE at the scan rate of 50 mV/s and potential range from − 0.25 V to + 0.6 V for 15 cycles. 15 cycles of CV were repeated to achieve stability and reproducible curves. In the first few cycles, peaks were not differentiable since the partial binding of MIP over PdNPs decorated PEDOT/Au-SPE was noticed. The enhancement of peaks was observed during this repeated cycling process, confirming that the process of electro-deposition on the Au-SPE electrode did occur.

Electrochemical characterization of MIP_BabA/PdNPs/PEDOT onto Au-SPE

The electron transfer characteristics and performance of the modified electrode were evaluated by CV and EIS study in 5 mM Fe[(CN)_6]^3−/4− electrolyte. The CV graphs in the potential range of − 0.4 V to 0.6 V at a 50 mV/s scan rate of the modified electrode are shown in Fig. 5A. Compared to the bare Au-SPE (black) electrode, the anodic current response of PdNPs/PEDOT/Au-SPE (red) was increased several times due to the conducting nature of the nanomaterials. The electrodeposition of NIP and MIP_template showed a decrease in peak current with respect to MIP_BabA. This is due to the polymer’s occupied electrode surface, which restricts the current flow. On the basis of these variations, we can infer that MIP_template was successfully immobilized due to the decorated PdNPs/PEDOT surface. Though lowering of the current peak after ^BabAAg interaction was explained by the formation of the electron blocking layer, which hinders the electron transfer between the electrolyte and interface [28]. The electrodeposited MIP_template was then washed 3 times with an acetone and methanol solution to remove the template from MIP. Template removal enhanced the electron flow due to increased surface area on the electrode surface. Furthermore, the anodic current response of ^BabAAg@MIP_BabA/PdNPs/PEDOT/Au-SPE (^BabAAg@MIP/Pd-PEDOT) and ^BabAAb-^BabAAg@MIP_BabA/PdNPs/PEDOT/Au-SPE (^BabAAb-Ag@MIP/Pd-PEDOT) was increased and showed a peak-separation potential of them are 0.085 V and 0.11 V, respectively. This response indicates the condition for quasi-reversible behaviour of the system [29].

Fig. 5.

A CV curves and B Nyquist Plot with circuit diagram in 5 mM [Fe(CN)₆]^3−/4− electrolyte at 50 mVs⁻¹ of bare Au-SPE (black), PdNPS/PEDOT/ Au-SPE (red), NIP/PDNPs/PEDOT/ Au-SPE (blue), MIP_template/PDNPs/PEDOT/ Au-SPE (magenta), MIP_BabA/PDNPs/PEDOT/ Au-SPE (green), ^BabAAg@MIP_BabA/PDNPs/PEDOT/ Au-SPE (cyan), ^BabAAb -^BabAAg@MIP_BabA/PDNPs/PEDOT/ Au-SPE (yellow)

Figure 5B shows a typical Nyquist plot of impedance spectra of the modified electrode fabricated in the presence of 5 mM Fe[(CN)₆]^3−/4− electrolyte. Electrochemical impedance spectroscopy (EIS) measurements were carried out using the Bio-Logic SP-200 electrochemical workstation in the presence of 5 mM [Fe(CN)₆]³⁻/⁴⁻ prepared in 0.5 mM sodium phosphate buffer (pH 7.0). Impedance spectra were recorded over a frequency range of 100 kHz–0.1 Hz using an AC sinusoidal perturbation amplitude of 10 mV at the open circuit potential (OCP) as the DC bias potential. A total of 10 points per decade were collected for each measurement. The obtained spectra were analyzed using EC-Lab® Z Fit software with an appropriate equivalent circuit model. In support of CV graphs, impedance spectra of bare Au-SPE also exhibited a maximum R_ct value (679.25 Ohm) in comparison to PdNPs/PEDOT (276.67 Ohm). The MIP_template deposition at PdNPs/PEDOT/Au-SPE showed a relatively higher electron transfer resistance of R_ct (479.38 Ohm), which can be attributed to the lower conductivity of the fabricated electrode as compared to NIP (456.73 Ohm). Furthermore, after elution of ^BabAAg from the imprinted polymer platform, resistance again decreases with an Rct value of 408.74 Ohm, which showed the increase in conductivity of the electrode. The generated BabA-specific cavities serve as a channel for the diffusion of electrolytes within the coated MIP surfaces. After the addition of ^BabAAg@MIP/Pd-PEDOT, the conductivity was decreased, which was confirmed by an increased R_ct value of 523.87 Ohm, which further decreased to 388.36 Ohm after the reaction of ^BabAAb-Ag@MIP/Pd-PEDOT. The EIS spectra were fitted using EC-Lab’s Z Fit-Biologic software with an equivalent circuit, as seen in Fig. 5B. Overall, the Nyquist plots exhibited a semicircular region at higher frequencies, corresponding to the charge transfer process, followed by a linear region at lower frequencies associated with diffusion of the redox probe. The impedance data were interpreted using the equivalent circuit model shown in Fig. 5B, consisting of solution resistance (R1), interfacial capacitance (C2), charge transfer resistance (R2), and Warburg diffusion element (W3). The observed changes in semicircle diameter after successive electrode modification, template removal, antigen rebinding, and antigen–antibody interaction confirm the successful stepwise fabrication of the sensing platform and the corresponding modulation of interfacial electron transfer properties.

Detection strategy of MIP_BabA modified electrode

In order to develop a sensitive immunosensor for H. pylori-specific BabA determination, a series of experiments was performed to optimize the best environmental conditions, such as concentration, pH, temperature, and storage stability for the fabricated sensor. To ascertain the applicability of the designed immunosensor for quantitative analysis of BabA protein, the performance of the fabricated electrode in the concentration range of 0.1–50 ng/ml was investigated by CV measurements under optimum conditions. In clinical samples, the absolute protein concentrations of important virulence factors or adhesins of H. pylori have not been measured. Protein-level measures are not reported in the literature currently under publication; instead, it solely discusses seropositivity, gene prevalence, or relative expression. Although literature reports a VacA biosensor with a range of 0.1–12.8 ng/ml. Prior research on related bacterial proteins (such as CagA and VacA) and serum antigens in infectious disorders usually falls between sub-nanograms to tens of ng/mL [30, 31]. As shown in Fig. 6A, the MIP/Pd-PEDOT/Au-SPE current response decreased with increasing ^BabAAg concentration, indicating that the imprinting pores were occupied by ^BabAAg. Once the maximum binding of ^BabAAg (50 ng/ml) was observed over the MIP electrode, the electrode was used for ^BabAAb detection. The CV graphs of ^BabAAb-Ag@MIP/Pd-PEDOT are shown in Fig. 6B, which reveals increased peak current with increasing ^BabAAb concentration from 0.1 to 50 ng/ml. The linear relationship was obtained between peak current and ^BabAAg concentration from 0.1 to 50 ng/ml with regression equation I(mA) = − 0.060 ^BabAAg (ng/ml) + 0.793 and a correlation coefficient of 0.964, as shown in Fig. 6C. Furthermore, the linear relationship was also observed for peak current in response to ^BabAAb concentrations from 0.1 to 50 ng/ml with regression equation I(mA) = − 0.094 ^BabAAb (ng/ml) + 0.714 and correlation coefficient of 0.958, as shown in Fig. 6D. This explains the antigen–antibody interaction which have taken place at the surface of the modified electrode. The sensitivity of ^BabAAg@MIP/Pd-PEDOT and ^BabAAb-Ag@MIP/Pd-PEDOT was calculated as 0.060 and 0.094 mA/ng/ml, respectively, using log concentration and current response; sensitivity of ^BabAAg@MIP/Pd-PEDOT and ^BabAAb-Ag@MIP/Pd-PEDOT was calculated as 0.002 and 0.003 mA/ng/ml using concentration and current response. The determination of recovery rate, concentration of 2.5 ng/ml, was tested on ^BabAAg@MIP/Pd-PEDOT, where observed accuracy was 93.6%. Additionally, the selectivity coefficient for MIP and NIP binding for both platforms was 3.61 and 3.73, respectively. With coefficients of 3.61 and 3.73, both platforms showed similar selectivity. While the slightly lower coefficient for the NIP confirms minimal non-specific binding, these values show that the MIP showed preferential binding toward the target analyte. Overall, the findings support the MIP platform's recognition sites’ reliability and imprinting effectiveness.

Fig. 6.

Voltammetric responses of fabricated electrode ^BabAAg@MIP/Pd-PEDOT with A different ^BabAAg concentrations, B different ^BabAAb concentrations. The standard calibration curve between C log of ^BabAAg concentration, and current response, D log of ^BabAAb concentrations, and current responses, in 5 mM [Fe(CN)₆]^3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.4) at 50 mVs⁻¹

Influence of scan rate on the fabricated electrode

The scan rate can control the redox mechanism of action of deposited compounds onto the working electrode. Therefore, to examine the scan rate effect on the fabricated electrode, voltammetric studies were performed in 5 mM Fe[(CN)₆]^3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.5). Initially, current responses of ^BabAAg@MIP/Pd-PEDOT (Fig. 7A) and ^BabAAb-Ag@MIP/Pd-PEDOT (Fig. 7B) were studied at different scan rates ranging from 10 to 120 mV/s in the presence of 0.1 ng/ml ^BabAAg and the potential ranges from − 0.4 V and + 0.6 V, as shown in Fig. 7A, B, respectively. The results from these studies revealed a quasi-reversible process that demonstrates the diffusion reaction for electron transfer. From 10 to 120 mV/s scan rates, symmetrical redox peaks were obtained with relatively similar potential responses. Moreover, the current responses were increased with the increase in scan rate ranges, which can be seen in peak-to-peak separation. The clear oxidation peaks prove the steady electron transfer on the electrode surface.

Fig. 7.

Voltammetric responses of current vs. potential, for A ^BabAAg@MIP/Pd-PEDOT and, B ^BabAAb-Ag@MIP/Pd-PEDOT on various scan rates, 0.5 mM [Fe(CN)₆]^3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.4) with 0.1 ng/ml BabAAg. The standard calibration curve between sensor responses and the square root of scan rate in 5 mM [Fe(CN)₆]^3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.4) at 50 mVs⁻¹, for C ^BabAAg@MIP/Pd-PEDOT and, D ^BabAAb-Ag@MIP/Pd-PEDOT

It shows a linear increment in cathodic and anodic peak currents with the square root of scan rate (Fig. 7C, D), with the following regression equations:

This data illustrated that the electrocatalytic reactions of the fabricated electrode are a controlled diffusion process. The scan-rate experiment used the redox probe of [Fe(CN)₆]^3−/4− in the electrode interface to determine the electron-transfer mechanism. A linear relationship between the maximum current amplitude and the square of the scan rate appears hence showing that the electron-transfer reaction of the redox probe is diffusion-controlled [32]. It is important to note that the target analyte (BabAAg) is non-electroactive and does not undergo direct redox reactions at the electrode surface. Instead, BabAAg is recognized through specific non-covalent interactions within the imprinted cavities of the MIP layer, which can be considered as a surface-confined adsorption-like process [33]. This adsorption of the analyte within the MIP matrix modulates the accessibility of the redox probe to the electrode surface, thereby altering the electron transfer process. Such behaviour is characteristic of indirect electrochemical sensing mechanisms, where the signal originates from the diffusion of the redox probe rather than the analyte itself [34].

The elaborated illustrations of Ip vs. ν plot and log Ip vs. log ν plot are given in Fig. S3. The plots of Ip versus ν (Fig. S3A, B) show that there is strong linear correlation which reflects processes related to the electrode surface. To further understand the charge transfer mechanism, the log Ip versus log ν plots (Fig. S3C, D) had been drawn and the results acquired a very good linearity with slope coefficients of about 0.636 and 0.341 respectively. Traditionally, a slope value close to 0.5 is that of a diffusion-controlled process, whereas the slope value close to 1.0 indicates an adsorption-controlled process. The determined slopes, being nearer to 0.5 but still not ideal, allude that the electrochemical reaction would be majorly diffusion-controlled and, at the same time, a partial role played by adsorption with reference to charge transfer, consequently suggesting a mixed charge-transfer reaction.

Effect of pH value, temperature, and stability on the binding capacity of ^BabAAg@MIP/Pd-PEDOT & ^BabAAb-Ag@MIP/Pd-PEDOT

The influence of pH and temperature on the redox behaviour of the fabricated electrode was examined in the presence of 0.1 ng/ml ^BabAAg in 5 mM Fe(CN)₆^3−/4− electrolyte. As shown in Figs. 8A, B, redox peak current was decreased from pH 6 to pH 7.0 and then gradually increased till pH 7.8 at ^BabAAg@MIP/Pd-PEDOT. There was a gradual decrement in peak current from pH 6 to pH 7.0 in the case of both ^BabAAg@MIP/Pd-PEDOT and ^BabAAb-Ag@MIP/Pd-PEDOT, which is due to the strong binding or optimum binding of Ag-Ab over the working electrode. Furthermore, the electrochemical response was increased (due to the decrease in Ag-Ab binding) while there was an enhancement in the pH value up to pH 7.8. It is because of the denaturation of Ab and loss of its activity or stability due to the extremely acidic or basic surroundings. The pH values confirm that the minimum current response, correspond to maximum analyte binding, was obtained near neutral pH conditions. A detailed representation of the numerical data is provided in the Supplementary Information (Table S3). pH 7 was chosen as the most favourable pH for the present electrode. pH 7 was selected as the optimal condition, as it provides a balance between strong analyte binding and physiological relevance. Furthermore, pH values above 7.8 were not explored, as extreme alkaline conditions may affect the structural stability of biomolecules and disrupt specific interactions within the MIP matrix.

Fig. 8.

Voltammetric responses of, A ^BabAAg@MIP_BabA/PdNPs/PEDOT/Au-SPE and, B ^BabAAb and ^BabAAg@MIP_BabA/PdNPs/PEDOT/Au-SPE on various pH in 0.5 mM [Fe(CN)₆]^3−/4− electrolyte with 0.1 ng/ml ^BabAAg

As MIP polymer is thermostable and antibodies are temperature sensitive, therefore the temperature-based study is useful in developing a highly sensitive electrochemical sensor. As shown in Fig. 9A, for ^BabAAg-platform, there was an increase in peak current from 20 to 35 °C, the current increased from 0.69 mA at 20 °C to 0.76 mA at 35 °C, followed by a further increase to 0.82 mA at 40 °C.

Fig. 9.

Voltammetric responses of, A ^BabAAg@MIP_BabA/PdNPs/PEDOT/Au-SPE and, B ^BabAAb and ^BabAAg@MIP_BabA/PdNPs/PEDOT/Au-SPE on different temperatures in 0.5 mM [Fe(CN)₆]^3−/4− electrolyte containing 0.5 mM sodium phosphate buffer (pH 7.4) with 0.1 ng/ml ^BabAAg

Unlike this, ^BabAAb-^BabAAg-platform, peak current increased gradually with increasing temperature from 10 to 50 °C (Fig. 9B). The study showed that from 20 to 35 °C, there is a possibility for optimal binding of antigen within the MIP_BabA, and after that current response was decreased. However, in the case of Fig. 9B, an antigen–antibody interaction was taking place. These observations support that optimal binding conditions are achieved around physiological temperature (~ 35 °C), while higher temperatures may affect biomolecular interactions. A detailed representation of the numerical data is provided in the Table S4.

Spiked sample analysis in serum

In order to estimate the presence of H. pylori using ^BabAAg@MIP/Pd-PEDOT and ^BabAAb-Ag@MIP/Pd-PEDOT for the clinical samples, 10 human serum samples, were obtained from the Bio-Diagnostics Lab located at Rohini, New Delhi. 1 ng/ml spiked concentrations of ^BabAAg were analyzed 5 times in human serum samples, and the relative standard deviation (RSD) was calculated to determine the precision of the sensor. The RSD and the average recovery were calculated as 3.1% and 94.8% (Table S1). The accuracy test was performed for inter-batch and intra-batch coefficients of variation by adding serum samples in a gap of a week. The values were calculated as < 4.98% and < 5.67%, respectively. A comparison of the analytical performance of various H. pylori sensing platforms is given in Tables 2 and 3 [29–31, 35–39]. The reliability and accuracy of the developed immunosensor for BabA detection was evaluated by comparing its performance with the conventional ELISA assay (Table S1). The high degree of linearity (Fig. S2) indicates that the immunosensor can be used as a reliable, rapid, and cost-effective alternative to ELISA for BabA detection, with comparable accuracy, but with the advantages of simplicity and potential for point-of-care applications.

Table 2.

A comparison of the analytical performance of various H. pylori sensing platforms

Sensor type	Biomarker	Detection limit	Linear range	References
Microfluidic	H. pylori antigen	1*104 ng/ml	1*104–1*106 ng/ml	[35]
Microfluidic	CEA PGII	0.37 ng/ml 2.5 ng/ml	0.37–90 ng/ml 2.5–80 ng/ml	[36]
Piezoelectric	H. pylori antigen	1*105 ng/ml	1*105–2.5*105 ng/ml	[37]
Microfluidic	H. pylori Antigen–antibody	1*102 ng/ml	1*102–1*10 5 ng/ml	[38]
Electrochemical	CagA	0.2 ng/ml	0.2–50 ng/ml	[39]
Colorimetric Electrochemical	DNA DNA	104 cfu/ml 0.15 nM	NR 0.3 nM–0.24 μM	[40] [41]
Microfluidic	H. pylori Antigen–antibody	0.37 U/ml	0–100 U/ml	[42]
Electrochemical (MIP)	BabA antigen/ antibody	0.1 ng/ml	0.1–50 ng/ml	This work

Table 3.

A comparison of the H. pylori sensing platform with existing analytical methods [43, 44]

Detection method	detection limit	Time to result	Complexity/cost	Invasiveness
Proposed sensor	0.1 ng/mL	~ 2 min	Low (portable device)	Invasive
ELISA	~ 1 ng/mL	2–4 h	Moderate	Non-invasive
PCR	~ 10–100 copies	4–6 h	High (lab-based)	Invasive
Urea breath test (UBT)	Indirect detection	~ 30–60 min	High (special equipment)	Non-invasive
Stool antigen test	Moderate	1–2 h	Moderate	Non-invasive

Selectivity study

To evaluate the selectivity of the developed sensor, the response toward BabA was compared with several potentially interfering biomolecules, including CagA, VacA, AFP, BSA, glucose, and ascorbic acid. The relative response of BabA was established at 100% while other analytes produced responses between 9 and 15% as shown in Fig. 10. The sensor shows high recognition capability toward BabA with negligible cross-reactivity because BabA stands out from the other tested molecules. The low glucose and BSA response values stem from non-specific binding, but these values remain below the specific BabA response. The imprinted sensing platform shows high selectivity and specificity toward the target analyte because it detects the target analyte reliably even when interferents have similar structures or chemical properties. Also, the biosensor demonstrated high precision, with an RSD of ~ 1%, confirming the reproducibility of the measurements. The fabricated sensor was found to be highly specific as it showed no significant response with other interfering compounds. The shelf-life of the ^BabAAg@MIP/Pd-PEDOT and ^BabAAb-Ag@MIP/Pd-PEDOT was examined via a cyclic voltammetric current response in the presence of 0.1 ng/ml standard ^BabAAg solution containing PBS at an interval of 7 days for 2 months. It’s pragmatic that developed electrodes keep hold of the activity up to 90% for 7–8 weeks when stored at 4 °C. All the experiments in this study were repeated three times, and three numbers of electrodes were used to check the stability of the experimental parameters. The sensor response of interferents for ^BabAAg@MIP/Pd-PEDOT is give in Table S5.

Fig. 10.

Selectivity study: Interferents CagA, VacA, AFP, BSA, glucose, and ascorbic acid were selected for the selectivity study, producing biosensor responses below 15%, indicating high sensor specificity for BabA detection

Conclusion

For the first time, we have reported the imprinted polymer-based detection platform for ^BabAAg and associated antibodies generated against H. pylori infections. The commercially available Au-SPEs were used because of their small portable size, low cost, and requirement for fewer reagents. ^BabAAg was allowed to interact with a fabricated sensing electrode surface which have ^BabAAg-specific cavities formed after the removal of the template from MIP matrices. The MIP sensor selects BabA through molecular imprinting because this process creates template-specific cavities during polymerization. The cavities in the polymer structure match BabA dimensions and functional group positions, which enables specific binding. The imprinting process utilized functional monomers that selected key BabA residues through hydrogen bonding and electrostatic and hydrophobic interactions. The imprinting effect establishes specificity, but researchers recognize that small non-specific interactions, such as van der Waals forces or non-covalent adsorption on the polymer surface, could also affect the binding process. Future research could include control protein tests and competitive binding studies to precisely determine the role of non-specific interactions. The experiments were designed in such a manner that the fabricated sensing electrode would detect the ^BabAAg in clinical samples, and then the same platform could be used for antibody detection, also to cross-verify the findings. The fabricated biomimetic polymeric system exhibited excellent selectivity and specificity towards H. pylori-specific ^BabAAg, which has been confirmed by detecting ^BabAAb on the same platform and through observation of its activity in the presence of various interferents. The stability of the fabricated system was observed by a scan rate study. The scan rate can control the redox mechanism of action of deposited compounds onto the working electrode. Relatively, the wide range of linearity has been observed for analyte binding in the studied concentration range of 0.1–50 ng/ml, and high detectability by LOD of 0.1 ng/ml. The other optimum conditions, namely pH, temperature, and scan rate, were observed thoroughly. The developed sensor is highly accurate due to the dual mode of detection property in human serum samples with RSD and the recovery of 3.1% and 94.8%, respectively. Thus, the facile detection property of the developed electrochemical sensor is prone to sensitive monitoring of H. pylori-induced gastric carcinoma and other associated gastric diseases. Future work will focus on performing cross-reactivity studies with structurally similar H. pylori adhesins such as SabA, HopQ, and others to validate the molecular selectivity of the developed MIP sensor. The research on BabA-imprinted electrochemical sensor development and characterization serves as the main focus of this study, and it provides essential groundwork for future lab-on-a-chip implementation. The study did not investigate multiplexing capabilities, miniaturization, or portability, but these features represent the planned development stage. The upcoming research would concentrate on sensor integration and simultaneous detection of multiple H. pylori virulence factors and wireless or smartphone-based reading systems. The development of such advancements would enable the creation of a complete point-of-care diagnostic device with improved clinical applications. The current experiment used cyclic voltammetry as the electrochemical approach to study interfacial electron transfer behaviour and authenticate the sensing principle of the developed MIP-based electrode. CV is generally recognized as a required instrument in the investigation of redox processes, surface alteration and charge-transfer properties in electrochemical sensors, especially in early sensor design. It provides a full listing of facts on reversibility and electrode kinetics [34]. Moreover, a number of studies on MIP-based electrochemical sensors have also taken advantage of CV to evaluate sensor behaviour as well as ensuring successful fabrication and interaction with the analyte. However, it is also appreciated that the techniques like the differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) offer much better sensitivity and better signal resolution making it more suitable in precise analytical quantification. These sophisticated methods will be explored in future studies in order to enhance performance in detection and reduce the limit of detection of the sensor being developed.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

Au-SPE

Screen printed gold electrode

PEDOT

Poly(3,4-ethylenedioxythiophene)

PdNPs

Palladium nanoparticles

NIP

Non-imprinted polymer

MIP

Molecularly imprinted polymer

^BabAAg

BabA specific antigen

^BabAAb

BabA specific antibody

MIP_template

MIP with template

MIP_BabA

MIP without template

MIP_BabA/PdNPs/PEDOT/Au-SPE

Modified sensing electrode

^BabAAg@MIP_BabA/PdNPs/PEDOT/Au-SPE

BabA antigen bound with BabA-specific MIP

^BabAAb-^BabAAg@MIP_BabA/PdNPs/PEDOT/Au-SPE

BabA antibodies interaction with BabA Ag on modified sensing electrode

Author contributions

Authors’ contribution: Project administration; Dr. Nidhi Chauhan and Dr. Utkarsh Jain; Conceptualization; Nidhi Chauhan, Kirti Saxena, and Smriti Gaba; Writing—review and editing: Smriti Gaba and Shringika Soni; Investigation; Kirti Saxena and Shringika Soni; Methodology; Nidhi Chauhan; Supervision: Dr. Utkarsh Jain, Bansi Dhar Malhotra; Validation: Dr. Nidhi Chauhan.

Funding

Science &amp; Engineering Research Board, SB/DF/011/2019, Department of Science and Technology, DST/WOS-B/HN-12/2021

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

Ethical approval for this study was obtained from the Institutional Ethics Committee (IEC), Amity University, Uttar Pradesh, Noida (Ref No: AUUP/IEC/APR/2022/9).

Consent to participate

Written informed consent to participate in this study was obtained from all human participants/donors involved in the study.

Consent for publication

Written informed consent to publish was obtained from all participants involved in this study. The participants voluntarily agreed to the use and publication of their data for research purposes. All procedures performed in this study involving human participants were conducted in accordance with the ethical standards of the institutional ethical committee.