Amine functionalized transition metal dichalcogenides based nanoparticles towards label-free immunosensing platform for antibiotics detection in food samples

Abstract

The World Health Organization (WHO) has been reported that antimicrobial-resistant (AMR) bacteria have become the basic risk to human health caused due by declining potency and excessive use of antibiotics [1, 2]. During clinical/veterinary practice, antibiotics have selective pressure on bacteria that speeds up the bacterial development having AMR properties owing to pharmaceutical accumulation in the water systems and food chains. The annual mortality rate related to infections from antibiotic-resistant bacteria is as much as nearly 23,000 deaths in the U.S., according to a report of the Centers for Disease Control and Prevention [3]. Recent studies have revealed that antibiotics with enormous concentrations are found in India's drinking water, surpassing the maximum regulatory limits (MRLs). This has led to a severe effect on susceptible residents existing close to industrial services, as nearly 58,000 new-borns die in these areas of India from multi-drug resistant infections [4-6]. The tetracyclines, sulfonamides, macrolides, fluoroquinolones, and β-lactams classes of antibiotic compounds are often present in aquatic environments [7]. Ampicillin (AMP) is one of the most used b-lactam antibiotics, a water-soluble chemical substance. It is extensively used in agriculture, livestock, poultry, aquaculture, veterinary, and human medicine to prevent and treat contagious diseases [8] due to the remarkable destroying ability for gram-negative and positive bacteria [9]. The overdose of antibiotics or antibiotic residues can cause severe environmental and food safety concerns that triggered various human and animal health complications in past decades involving endocarditis, membranitis, intestinal infection, and irritability [8, 10]. Therefore, detecting AMP residues in foodstuffs is essential for protecting human health as it can lead to adverse reactions, comprising anaphylactic disorders. Hence, there is an urgent need to develop reliable, sensitive, portable, inexpensive, and fast detection methods for detecting AMP, specifically.

Numerous commercial diagnostic tests are currently available for antibiotics in food samples and environments, either unable to detect antibiotic compounds selectively or need a lengthy response time (several hours) and offer semi-quantitative information only [11]. Over the decades, several attempts have been tried for developing various effective techniques for the detection of AMP residues in waters and agricultural products, which includes Raman spectroscopy [12], high-performance liquid chromatography (HPLC) [13], liquid chromatography-mass spectrometry/ mass spectrometry (HPLC-MS/MS) [14], surface plasmon resonance (SPR) [15], spectrophotometry [16], colorimetric methods [17], electrochemiluminescence [18], enzyme-linked immunosorbent assay (ELISA) [19], nuclear magnetic resonance [20], and immunochemical detection [21]. Such methods are sensitive to AMP detection, but due to high cost, sophisticated instrumentation, complicated sample preparation, and complex instrument operation, it restricts its practical applications for the on-site assays. Therefore, simple, rapid, miniaturized, and low-cost electrochemical immunosensor hold considerable attention for detecting antibiotics [22, 23]. By combining the high selectivity of antibodies with the highly sensitive transducers, antibodies-based sensors, i.e., immunosensors, have become an effective analytical technique for antibiotic residues determination [24, 25]. Also, antibody usage enhances the efficiency of the immunochemical response due to faster assay kinetics along with an increased surface area and also minimizes matrix effect with improved washing and separation steps [26, 27].

To address the antibiotic resistance problem, here we aimed to fabricate a label-free immunosensing platform for the first time based on nanostructured molybdenum disulfide nanoparticles (nMoS₂ NPs) deposited on Indium tin oxide for the electrochemical detection of Ampicillin (AMP). The stable and high surface area nMoS₂ NPs were made by a low-temperature one-step hydrothermal route, bestowing the carrying capacity of monoclonal antibodies (anti-AMP) through an amide linkage. Bovine Serum Albumin (BSA) was utilized to block the non-specific sites on the immunoelectrode surface. The spectroscopic, morphological, and structural characterization of the proposed electrodes were performed using various analytical techniques like X-ray diffraction (XRD), Raman spectroscopy, Fourier transform-infrared spectroscopy (FT-IR), Field emission-scanning electron microscopy (FE-SEM), Transmission electron microscopy (TEM), Atomic force microscopy (AFM) and electrochemical techniques such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The DPV method was utilized to evaluate anti-AMP and AMP interaction on the electrode surface.  The change in the MoS₂ interface current was caused by the specific binding of AMP to the antibodies immobilized onto the nMoS₂ NPs/ITO electrode. Other antibiotics such as norfloxacin, ofloxacin, and ciprofloxacin were used in this experiment to test the specificity of the immunosensors. The developed immunosensor exhibits high sensitivity [41.81 mA (log mg mL^-1)^-1], a broad detection range (0.0325-64 mg mL^-1) having a significant detection limit (0.028 mg mL^-1) towards detection of AMP having excellent selectivity, acceptable stability, and reproducibility. Furthermore, the applicability of the proposed immunosensor was tested in spiked milk, water, and orange juice, and the results confirmed the consistency of the immunosensor. This work shows the future potential of nMoS₂ NPs in developing immunosensors for sensitive detection of antibiotics and other analytes with low cost that might prove beneficial for the on-site detection of chemical or biological molecules.