There is increasing interest in the application of biosensors in the rapid detection of biomarkers to provide point-of-care (POC) testing [1, 2]. A biosensor is made up of a biorecognition (biocomponent or bioreceptor) unit, a signal transducer unit, and a unit that relays and processes information into electrical signals. They are potable, easy to use, and have a low manufacturing cost [3–5]. Clinical diagnosis of cancer requires detection tools that are rapid, sensitive and specific; therefore new cancer diagnostic tools are exploring the development of biosensors for these desirable attributes compared to conventional techniques used in the field of cancer diagnostics. The recognition element determines the selectivity of the biosensor by recognising the bioreceptor to produce a physiochemical signal, whereas the transducer determines the sensitivity of the biosensor by converting the biological response generated by the interaction of biorecognition molecules (cells, antigens, enzymes, antibodies, DNA, and RNA) with the analyte into a measurable electrical signal [4, 6]. Biorecognition molecules are immobilised to serve as a bridge between the recognition unit and the transducing surface. Some of the biorecognition molecules require immobilising materials to help increase the electrochemical reactivity of a transducer, as they help the biorecognition molecules to attach well to the transducer surface for energy transfer [7, 8].

Cytosensors are cell-based biosensors that can make use of electrochemical techniques in their detection routes. The electrochemical cell-based detection system promises to be very sensitive and exhaustive, as it gives great details of functional information about the target analyte [9]. A significant advancement in biosensing is the development of an ultra-high-resolution biosensor that utilises reduced graphene nanowalls. The biosensor shows promise as an efficient, label-free sensor for detecting single DNA nucleotides [10]. However, one of the major challenges with electrochemical biosensors is the detachment of biomolecules from the transducing surface, which slows electron movement. The biosensor produces a weak biological signal limits the linear range of detection, hence hampering its sensitivity [7, 11]. Biomaterials such as graphene [12], chitosan [13], collagen [14], conductive polymers [poly(3,4-ethylenedioxythiophene) (PEDOT)] [15], and other inorganic materials [16] are used as immobilising materials in cancer biosensing to curb detachment problems; however, the immobilizing properties of a hydroxyapatite (HAp) biomaterial and PEDOT are accessed in this study. HAp [Ca₁₀(PO₄)₆(OH)₂] is a naturally occurring inorganic material derived from bones and teeth. HAp can also be synthesised from shells, corals, and calcium apatite or calcium phosphate precursors. The structural and chemical characteristics of HAp makes it biocompatible and diverse in applications. For biomedical purposes, it is used in drug delivery systems, bone tissue scaffolding, bone and tooth filling, and coating implant devices [17]. HAp has been widely used as a biomaterial as it does not stimulate the immune system directly and does not cause any systemic toxicity if reabsorbed. It has the capacity to inhibit cancer cell proliferation and induce apoptosis in several cancer cells, including osteocytes, colon cells, breast cells, and liver cells while sparing their normal cells respectively [17, 18].

The study investigates the electrochemical property (EP) of snail shell HAp (SHAp)/PEDOT: poly-4-styrene sulfonate (PEDOT: PSS) composite immobilising material for cytosensing, as illustrated in Figure 1. The SHAp material is synthesised from Achatina achatina snail shells and a phosphate-containing solution. PEDOT: PSS was added to improve the conductivity and adhesion of SHAp onto the electrode surfaces for cell immobilisation studies. The material was initially characterised using X-ray diffractometry (XRD), Fourier transform infrared (FTIR) spectroscopy, and Raman spectroscopy to determine its structural configuration. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) are electrochemical characterization techniques subsequently employed to access the EPs of the SHAp/PEDOT: PSS composite before and after cellular immobilisation.

The study aims to investigate the use of HAp as an immobilising material for label-free detection of cells. HAp was synthesised from Achatina achatina snail shells (SHAp) at 850℃ through a wet chemical precipitation method. Structural identification of the SHAp was performed alongside a CHAp using XRD, FTIR, Raman spectroscopy, and SEM. The XRD patterns of CHAp indicated only HAp phases, which confirmed that the CHAp is pure HAp. Also, the HAp and calcite phases present in the XRD pattern of SHAp confirmed that the synthesised material contained HAp and calcite. CHAp and SHAp were further proffered to be HAp molecules with the presence of PO₄^3–, CO₃^2–, and OH^– functional groups. FTIR and Raman spectroscopy predict CHAp and SHAp to be type B carbonate (CO₃^2–) substituted HAp, thus replacing the PO₄^3– functional group with CO₃^2–, resulting in a low OH^– transmission peak in FTIR and a high-intensity peak in the Raman spectroscopy. Type B carbonate (CO₃^2–) substituted apatite is mostly found in biological sources of apatite [21, 22]. The level and share of CO₃^2– substitution could be influenced by the source of HAp and the calcination temperature used. Snail shells naturally deposit carbonate materials in their shells due to their feeding habits; moreover, the calcination temperature of 850℃ helps to decompose the organic waste for purer calcite formation [17, 23].

The inorganic compound SHAp was exploited for the selective adsorption of cells in the biosensor. Transducers in electrochemical biosensors transform a chemical reaction occurring on the surface of the biological elements into an electrical signal. The SHAp material affords the necessary sensitivity to the sensor, as the signals produced must correlate directly to the signals measured. In this regard, electrochemical biosensors should possess good signal transduction abilities. The suitability of a carbon-based screen-printed electrode (SPE) as a base transducer in the sensor design was in PBS and ferrocyanide solutions. Carbon-based electrodes have a wide potential window and a low background current, making them preferable in the formation of SPE. SPEs come in a simple electrode setup and are cost-effective in mass production, which allows single usage for independent tests without contamination [24, 25].

SHAp is a suitable interface at which redox reactions occur because of the B-type carbonate substitution it underwent. The ionic diameter of carbonate is smaller than that of phosphate, so the replacement of the phosphate with carbonate allows SHAp to have a new structure conformation that makes SHAp more reactive than CHAp [21]. CV and EIS results suggest that SHAp is more conductive and introduces less impedance to the SPCE surface as compared to CHAp. Due to the improved electrochemical responses of the SHAp, it was selected as the suitable material for SPCE modifications in the study. For diagnostic purposes, a good biosensor should be able to produce sharp electrochemical responses; hence, PEDOT: PSS, a conductive polymer, was introduced into SHAp to form a SHAp/PEDOT: PSS composite. The use of composite that involving conductive polymers with other biomaterials has been widely used in biosensor fabrication, and the formation tends to sharpen and increase the electrochemical responses. In addition, conductive polymer composites tend to enlarge the surface area of immobilising materials in biosensors, marking them more sensitive and efficient in capture [25]. A very drastic increase in peak current was observed when PEDOT: PSS was introduced into the SHAp, indicating a sharp rise in conductivity as compared to the SHAp alone. This could be due to the synergistic increase in conductivity of the SHAp and PEDOT: PSS, thus a synergistic conductance from the SHAp and PEDOT: PSS. Impedance results complement the voltametric measurements and show that the introduction of PEDOT: PSS drastically reduced the impedance produced by the SPCE inherently and the SHAp. The introduction of the conductive polymer into SHAp not only increased its conductivity but also improved SHAp adhesion on the electrode surface as compared to SHAp alone for cell immobilisation studies. The high R² values obtained for the anodic peak (Ipa) and cathodic peak (Ipc) currents of the fabricated SHAp/PEDOT: PSS modified SPCE for both the oxidation and reduction cycles suggest that there is high consistency in current values when the scan rate is increased, and this indicates that the electron flow mechanism of modified SPCEs was in a diffusion-controlled state. The CV and EIS data provided show that SHAp/PEDOT: PSS is a good immobilising material for carbon-based electrodes for sensor development as it allows for good signal transduction by removing all inherent impedance from the transducing surface; hence, any signal obtained is from the biorecognition element immobilised on the transducing surface.

Human cells, both pathogenic and non-pathogenic, contain sialic acid, a carbon receptor molecule that can be found on the terminal regions of glycan chains on cell membranes. Sialic acid moieties have been associated with several diseases, including cancer, and find relevance in cell targeting. Sialic acid moieties are important in key activities including cellular signalling and proliferation, cell-to-cell adhesion and interaction, and host-to-pathogen interactions. HAp is known to have an adhesive interaction with sialic acid on cell surfaces, although this phenomenon has not been exhaustively understood yet [19, 26]. The detection of cells through the interaction of sialic acid on cell surfaces and SHAp can be used for label-free cancer cell detection. This would help in the reduction of interferences from normal cell activities, improvement in kinetic measurement, simplicity in assay formation, and a reduction in assay measurement time [27]. Cell lines (HEK 293T and PC 12) and normal cells (RBCs) were immobilised, and the CV recorded a high current for RBC as compared to PC 12 and HEK 293T, respectively. The CV results were complemented by EIS results, which further suggest that RBC recorded lower electrolyte impedance values than PC 12 and HEK 293T. These results could be attributed to the difference in the electrochemical behaviour of individual cells through polarisation and depolarization mechanisms resulting in an ion-concentration gradient across their cell membranes. Normal human cells mostly undergo polarisation by the efflux of ions from their cytoplasm to their outer environment to obtain a balanced resting potential, thereby increasing their external ion concentration, whereas cancer cells mostly undergo depolarization through the influx of ions from their external environment into their cytoplasm, thereby reducing the ion concentration in the outer environment [19, 28]. Ion concentration across cell membranes is reported to facilitate electron transfer. The electrochemistry of electron transfer along the electrode surface is altered as cells are immobilised on its surface. The electrodes face varying levels of resistivity from cells as charges flow towards the electrode surface. The electrolyte resistivity of cells differs as the types of polarised cells differ. Cancerous cells are reported to possess a net negative charge across their cell membranes, which contributes to the resistance to electron transfer along the electrode surface. On the contrary, RBCs are reported to have a net positive charge on the membrane, hence facilitating the flow of electrons along the electrode surface, resulting in the decreased R_E recorded [19, 28].

It is important to note that the SHAp/PEDOT: PSS modification results in an alteration of the surface structure of the SPCE. The asperity of the SHAp surface means cells can attach better without direct attachment to the electrode surface. The proposed attachment mechanism of cells to the SHAp on the SPCE surface is depicted in Figure 8A. Cells are captured within the porous matrix of the SHAp, allowing for direct and effective charge transfer between cells and the SPCE surface. This is in contrast with other studies [8, 29, 30] where cells lie directly on electrode surfaces, insulating the electrode surface from direct charge interaction with the biorecognition elements, as shown in Figure 8B.