Gold nanoparticle (AuNPs@rGO)-modified electrochemical immunosensor for early detection of Orientia tsutsugamushi-specific TSA antigens in typhus

Abstract

An electrochemical immunosensor device for the early detection of Orientia tsutsugamushi-specific TSA antigens, consisting of a carbon screen-printed paper electrode (SPPE) modified with AuNPs@rGO nanocomposites, an antibody immobilization interface and an electrochemical detection circuit.

Description

AREA OF INVENTION

[0001] The invention relates to biomedical diagnostic devices and biosensor technologies. In particular, it relates to an electrochemical immunosensor with gold nanoparticles stabilized on reduced graphene oxide (AuNPs@rGO) for the early detection of type-specific antigens (TSAs) of Orientia tsutsugamushi, the causative agent of spotted fever. The invention provides a portable, rapid, and cost-effective diagnostic platform suitable for resource-constrained and mobile healthcare applications. BACKGROUND OF THE INVENTION

[0002] Typhus, caused by Orientia tsutsugamushi, is a neglected tropical disease transmitted by the bites of the harvest mite, leading to severe fever and, if left untreated, multi-organ failure. Early and accurate diagnosis is crucial for effective treatment. However, conventional diagnostic methods such as serological tests (Weil Felix test, ELISA) and molecular methods (PCR) are often time-consuming, expensive, and require skilled personnel and appropriate laboratory infrastructure. This poses a significant challenge for rural and resource-poor regions where typhus is endemic. Recent advances in nanotechnology have enabled the development of electrochemical biosensors for rapid disease detection. These offer advantages such as portability, high sensitivity, and low power consumption. However, achieving high selectivity and signal amplification remains a major technical challenge.Gold nanoparticles (AuNPs) exhibit excellent biocompatibility and electron transfer capacity, while reduced graphene oxide (rGO) offers a large surface area and superior conductivity. Combining these materials can significantly enhance the performance of electrochemical immunosensors. Despite previous efforts, no system yet efficiently integrates AuNPs@rGO nanocomposites into a compact electrochemical platform specifically for the detection of O. tsutsugamushi-specific TSA antigens. Therefore, there is a need for a sensitive, stable, and field-deployable immunosensor for early detection that enables faster clinical decisions and reduces disease burden. SUMMARY OF THE INVENTION

[0003] The present invention provides an electrochemical immunosensor modified with AuNPs@rGO for the early detection of TSA antigens of the parasite Orientia tsutsugamushi. The system integrates a carbon-coated screen-printed paper electrode (SPPE) modified with AuNPs@rGO nanocomposites to improve electrochemical sensitivity and antibody immobilization efficiency.

[0004] The nanocomposite layer, produced using a single-step, low-temperature in-situ reduction process, offers a uniform and conductive surface that enhances signal transduction. The immobilization of anti-TSA antibodies on the AuNPs@rGO surface enables specific antigen-antibody interactions, generating measurable electrochemical reactions that correspond to the antigen concentration. This immunosensor provides a portable, rapid, and cost-effective diagnostic platform for the early detection of spotted fever in routine clinical practice and in the field.

[0005] The integrated design ensures high stability, reproducibility, and scalability. Manufacturing is simple, environmentally friendly, and suitable for mass production. The invention thus represents a significant advance in the field of biosensor-based diagnostics of infectious diseases. DETAILED DESCRIPTION

[0006] The invention describes an electrochemical immunosensor consisting of a carbon-printed paper electrode (SPPE), a modification layer made of AuNPs@rGO nanocomposite and a bioreceptor interface with immobilized anti-TSA antibodies.

[0007] The working electrode of the SPPE serves as an active sensor platform. The electrode surface is modified with AuNPs@rGO nanocomposites, which are synthesized via a one-step, low-temperature chemical reduction process. This process ensures the uniform distribution of the gold nanoparticles on the reduced graphene oxide matrix, resulting in improved conductivity and an increased surface area for biomolecule binding.

[0008] The nanocomposite synthesis system comprises a reaction chamber with temperature control and a stirrer to maintain a uniform reduction process. Gold ions are reduced in situ in the presence of graphene oxide and stabilizers, resulting in the uniform formation of AuNPs anchored to the rGO layers.

[0009] The electrode modification module includes a precision microdroplet dispenser that applies the AuNPs@rGO solution to the SPPE surface, forming a thin, conductive film upon drying. The modified electrode is then functionalized with anti-TSA antibodies via covalent or adsorption-based immobilization, creating a specific bioreceptor interface for O. tsutsugamushi TSA antigens.

[0010] The immunosensor's detection unit operates using electrochemical impedance spectroscopy (EIS) or differential pulse voltammetry (DPV). Antigen-antibody interactions alter the electrode's current response, enabling the quantification of antigen presence and concentration. The device also integrates a portable potentiostat, a microcontroller, and data processing software for real-time analysis.

[0011] The device housing is compact, battery-powered, and designed for mobile field use. It exhibits high selectivity for TSA antigens and minimal cross-reactivity with other bacterial proteins. Its analytical performance is characterized by high sensitivity, low detection limits, and fast response times (within minutes).

[0012] The sensor regeneration system enables multiple reuses through mild buffer washes to dissociate antigen-antibody complexes without damaging the nanocomposite surface. The design ensures stability over extended periods and is compatible with mass production using cost-effective materials.

[0013] This integrated electrochemical immunosensor platform offers a reliable diagnostic alternative to laboratory-based immunoassays and supports early disease management and outbreak monitoring in endemic areas.

Claims

[1] An electrochemical immunosensor device for the early detection of Orientia tsutsugamushi-specific TSA antigens, consisting of a carbon screen-printed paper electrode (SPPE) modified with AuNPs@rGO nanocomposites, an antibody immobilization interface and an electrochemical detection circuit. [2] Device according to claim 1, wherein the AuNPs@rGO nanocomposites are synthesized by a one-step in-situ low-temperature reduction process, resulting in uniformly distributed gold nanoparticles on a reduced graphene oxide matrix to increase electrical conductivity and surface area. [3] Device according to claim 1, wherein anti-TSA antibodies are immobilized on the electrode surface modified with AuNPs@rGO to enable specific antigen recognition and electrochemical signal transduction by means of impedance or voltammetry measurement. [4] Device according to claim 1, wherein the system comprises a portable potentiostat, a microcontroller-based signal processor and a display interface and enables fast, sensitive and cost-effective detection suitable for field and clinical diagnostic applications.