Development of innovative competitive amperometric immunosensors as promising tools in clinical diagnosis and food safety applications

The aim of the research project was the development of competitive amperometric biosensors for clinical diagnostics and food safety applications, as a part of a research activity focused on innovations in sensing systems investigated in our laboratories of analytical chemistry. In particular, the project purpose was the development of innovative sensors for detection of serum biomarkers for HIV (Human Immunodeficiency Virus) and HCV (Hepatitis C Virus) and the development of competitive immunosensor for detection of prolamins in food products for celiac disease patients. The number of subjects living with HIV/HCV co-infection is increasingly high, about 50000 in Italy, and this is mostly ascribable to the routes of transmission, which are common to the two viruses. For subjects who present one of the two infections, the co-presence has important consequences that can lead to a rapid course of the disease to the acute phase and, in some cases, to death; for these reasons, an early diagnosis through rapid and accurate diagnostic techniques is fundamental. The diagnostic methods currently available are mainly based on the detection of antibodies, viral nucleic acids or on the research of the capsid proteins. The first method is not useful for early diagnosis purposes, requiring a long period, called “window period”, for antibodies development by the immune system and their detection in blood stream. Methods based on the research of nucleic acids allow a more rapid diagnosis, although they require sophisticated instrumentation and expert technicians, in addition to high costs of analysis. A good compromise between the above-mentioned methods is the search of the viral capsid proteins, which are developed earlier than antibodies and are detectable in blood stream since the first weeks after the contraction of the virus, requiring easy-to-use instrumentations and inexpensive analysis. In the present study, we focus on these proteins, respectively p24 for HIV and NS4 for HCV, with the aim of developing immunosensors for early diagnosis of the infections, as alternative to the already available methods, mainly ELISA-based test (Enzyme-Linked Immunosorbent Assay). In a first stage of the research activity, the main purpose was the development of dual-chip devices aimed at detecting simultaneously the two capsid proteins, allowing the diagnosis of co-infection. Before using the dual chip device, HIV and HCV systems were studied individually, in order to assess the methods of detection for each specific antigen and to optimize the experimental conditions for each of them. The study was initially focused on p24 determination by means of amperometric immunosensors implemented on screen-printed electrodes. The most critical step in biosensors development is the functionalization of the electrode with the bio-receptor. This aspect required the study of different methodological approaches, based on the immobilization of anti-p24 antibodies or p24 antigen, for the realization of sandwich-type or competitive assays, respectively. In the case of the sandwich system, the easiest approach of functionalization evaluated was the direct incubation of anti-p24 antibodies on the working electrode of screen-printed glassy carbon substrates, functionalized with gold nanoparticles (GNP-SPCEs). The immobilization of anti-p24 antibodies evidenced critical aspects related to their proper orientation with consequent alteration of the binding properties for the target antigen. In order to allow the recognition of one or more epitopes of the corresponding antigen, the proper orientation of the antibodies can be induced promoting the linkage via the Fc fraction, using auxiliary proteins such as protein A and G. Although these proteins facilitate the correct orientation of the antibody, they do not ensure the stability of functionalization, as in the case of covalent linking. It was thus decided to evaluate the functionalization through covalent approach of Self-Assembled Monolayers (SAM), by using 11-mercapto-undecanoic acid, activated with N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide as coupling reagents. It was also experimented the combination of the SAM with the G protein, in order to ensure both the robust functionalization of the electrode surface and the correct orientation of the antibody. The best results were obtained using the competitive approach. Initially the immobilization of p24 antigen was performed both by direct incubation on the electrode surface using SAMs as linkers. In fact, although the direct electrode functionalization is the simplest approach, the chemisorption could involve functional groups which are fundamental for the immunochemical recognition of the antibody. To overcome this problem, a method involving the use of chitosan, as active layer immobilized on gold-free glassy carbon electrodes, in association to glutaraldehyde, as cross-linker agent, was successfully investigated. On the basis of these findings, it was realized a competitive amperometric sensor for p24 detection, based on its immobilization on screen-printed glassy carbon electrodes (SPCEs). The anti-p24 antibodies immunosorbed on p24-modified SPCEs were detected via alkaline phosphatase-labeled secondary antibodies, through an enzymatic reaction processed on hydroquinone diphosphate, used as substrate. In order to optimize the concentrations of antigen immobilized on the electrode surface and of anti-p24 antibody, a two factors and three levels experimental design was performed, working at a pre-fixed concentration of p24 in competition. A two-way analysis of variance (ANOVA) was applied to investigate the effects of the experimental parameters on the inhibition rate, taking place on the basis of the immunocompetition. The resulting interaction plot allowed us to individuate the optimal concentration of antigen on the electrode surface and antibody in competition, so that different concentrations of antigen in competition were explored. The outcome data were processed by a four- parameters logistic function, obtaining the corresponding inhibition curve. The developed sensor allowed to detect p24 at micrograms/mL levels, with good performance in terms of precision and trueness; however, since limits of detection and quantification were not adequate for diagnostic purposes, further optimization resulted necessary. Similar studies, still in progress, have also been performed with the aim of developing a similar competitive assay for HCV virus detection. To improve the performance of the sensor, in terms of diagnostic suitability for HIV infection, the use of single-walled carbon nanotubes (SWCNT) as electrode substrate was evaluated. These nanomaterials have the advantage of presenting a greater surface area, resulting in higher functionalization of the electrode, which is helpful to improve efficiency of electron transfer processes. Thus, starting from the optimal concentrations obtained with the previous experimental design, performance of new electrodes were evaluated, obtaining a remarkable improvement of the instrumental response. After a further optimization of the anti-p24 concentration, it was developed a sensitive device able to work in a lower concentration range, with limits of detection and quantification of 95 pg/mL and 2.6 ng/mL, respectively, more useful for diagnostic and clinical purposes. Concurrently with the study carried out individually on the two systems, the evaluation of dual device setup was performed. For this purpose, two elliptic working electrodes, sharing the same reference and counter electrodes, i.e. dual screen-printed electrodes were used. The main drawbacks when working with dual devices is the so called "cross-talk", taking place because of diffusion of the electroactive product of the enzyme reaction (hydroquinone) between the two working electrodes, giving rise to false positive results. This problem can be overcome with the use of the enzyme substrate 3-indoxyl phosphate (3-IP) in the presence of a silver salt (AgNO3). Alkaline phosphatase catalyzes the dephosphorylation of 3-IP, into a product able to reduce Ag+ to Ag0, which is localized where the enzymatic label AP is attached. The deposited silver can be electrochemically stripped into solution and measured by anodic stripping voltammetry, obtaining a signal related to the concentration of analyte to be detected. Concerning the determination of p24, also the dual devices requested optimization studies in terms of chitosan/glutaraldehyde, antigen/antibody concentrations and reaction times, in order to reach useful signals comparable with those obtained with the single SPCEs, working with hydroquinone diphosphate as enzyme substrate. However, preliminary studies with the dual device showed strong limits mainly ascribable to the size of the working electrodes and to their elliptical geometry, causing a great difficulty on their functionalization, with the risk of contamination and false positives, due the contiguity of the two electrodes. In order to further improve the performance of the immunosensors for determination of p24, a part of the research activity was carried out, within the context of a collaboration, with the Catalan Institute of Nanoscience and Nanotechnology (ICN2) in Barcelona (Spain), at the NanoBioelectronics and Biosensors Group, directed by Professor Arben Merkoci. The project carried out at ICN2 involved the use of magnetic beads (MBs) as substrates for immunoconcentration and bi-metallic nanoparticles as label enzyme-free agents for the electrochemical measurements. MBs were functionalized with p24, performing the subsequent antigen detection by using bi-metallic gold/silver nanoparticles, The electrochemical detection was carried out by exploiting the catalytic properties of bi-metallic nanoparticles, conjugated with the anti-p24 antibody, through direct and enzyme-free amperometric measurements. The integration of previous studies with those conducted during the period of stay at the ICN2 allowed to evaluate the advantages of sample pre-concentration on the magnetic beads, in perspective of immunosensor application in a complex matrix, such as blood serum. It was also possible to compare the performance of bi-metallic nanoparticles, as labeling systems alternative to enzymes. Another part of the thesis work was aimed at the application of the experimental protocols of the competitive assays for development and validation of an innovative amperometric immunosensor for the detection of gliadin and other allergenic prolamins, which have a great impact in the diet of celiac patients owing to their toxicity in celiac disease. It should be noted that the developed immunosensor is the first example of competitive device applied for detection of gliadin in food products. The novelty of this study relies in the assessment of the compatibility of the method with the use of complex solutions required for the extraction of the target analytes from food matrices, as raw materials or finished products, e.g. the Cocktail Solution®, composed by disaggregating and/or reducing agents, such as mercaptoethanol and guanidine, developed and patented by D.E. Mendez and approved by the Association of Official Agricultural Chemists, U.S. Department of Agriculture (AOAC). After optimization by means of experimental design procedures, the immunosensor was, tested for its applicability to the analysis of both raw materials and processed food. Limits of detection and quantification of 8 and 22 ng/ml of gliadin, respectively, were obtained in the case of ethanol exctracts of raw food materials. No interference from non-allergenic prolamins, such as those contained in corn and rice, typically used in the formulation of gluten-free foods, was observed. This is consistent with the special features of anti-gliadin antibody used in sensor development, attesting a further proof of the absence of non-specific responses. Good results were obtained also in the analysis of complex food samples, thus highlighting the good performance of the device as a powerful rapid and effective screening tool in quality control of gluten-free foodstuffs for celiac subjects.