Leishmaniases are a group of parasitic infectious diseases that can affect animals and humans and are transmitted by vector bite. There are two principal clinical forms are cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) [1]. In Brazil, the primary species responsible for CL include L. braziliensis, L. guyanensis and L. amazonensis, while VL is caused by L. infantum [2]. Their pathological manifestations differ in both clinical presentation and prognosis [3]. The VL is a globally distributed and re-emerging zoonosis, with domestic dogs serving as key reservoirs. Clinical signs of VL are nonspecific and may overlap with those of other diseases; some infected animals even remain asymptomatic, complicating laboratory diagnosis [4]. The spectrum of clinical presentation, from asymptomatic to the presence of all known signs, depends on the host immune response [5]. Diagnosis relies on clinical signs, parasitological, serological, and molecular methods. However, each approach has inherent advantages and limitations [1,[6], [7], [8]].

Parasitological methods remain the gold standard but are limited by their invasive nature, procedural complexity, and reliance on skilled personnel and infrastructure, making them less accessible in resource-limited settings [6,9]. Their sensitivity is further reduced in chronic or subclinical infections, and microscopic identification poses challenges due to morphological similarities with other trypanosomatids [7,10]. Molecular methods, while highly sensitive, demand specialized technicians, advanced laboratories, and stringent contamination controls, which are often impractical in endemic regions [1,7]. Serological assays, though non-invasive and rapid, face drawbacks such as cross-reactivity, inability to quantify infection load, and reagent stability issues [11]. To overcome these limitations, diagnostics aligned with the ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable) are urgently needed [1,6,7,12]. In this context, the integration of nanotechnology and biomolecular engineering offers a transformative platform for developing immunosensors that combine high sensitivity, miniaturization, and real-time biomarker monitoring, thereby advancing portable, cost-effective, and accurate diagnosis of Leishmania infections [5,13,14].

Immunosensors are widely utilized across diverse fields, such as clinical medicine, food safety, and environmental monitoring, and are increasingly adopted as diagnostic alternatives for various diseases [15,16]. These devices are solid-state affinity biosensors designed to detect specific targets (e.g., antigens or antibodies). Detection relies on the formation of a stable immunocomplex between the analyte and a surface-immobilized recognition element (antibody or antigen). This interaction induces a measurable signal at the sensor interface, which is then converted into a quantifiable output by a transducer [5,13,[17], [18], [19]]. Among these, electrochemical immunosensors emerge as a promising alternative, offering high sensitivity, rapid response, low cost, and compatibility with miniaturization for portable diagnostics [20,21]. Their versatility allows customization through diverse materials and fabrication approaches, including 2D [22,23] and 3D printing technologies [24,25]. Screen-printed electrodes, for instance, are widely used in immunosensor assembly, offering a robust platform for point-of-care applications [26].

The recombinant protein Lbk39, derived from the rk39 gene of L. braziliensis, shares 59 % amino acid identity with its homolog in L. infantum. When produced at a laboratory scale, it demonstrated sensitivity of 98 % (for human cutaneous leishmaniasis, CL) and 100 % (for visceral leishmaniasis, VL), with specificity of 88 % and 98 %, respectively [27]. This antigen was also evaluated for diagnosing canine visceral leishmaniasis (CVL), achieving 100 % sensitivity and 96 % specificity [28]. Given Lbk39's strong diagnostic performance, high sensitivity across leishmaniasis types, ease of production, and low cost, this study focused on constructing an immunosensor using screen-printed electrodes functionalized with recombinant Lbk39 as the antigen. The goal was to develop a cost-effective diagnostic device suitable for point-of-care applications.