Disposable electropolymerized molecularly imprinted electrochemical sensor for determination of breast cancer biomarker CA 15-3 in human serum samples.

Initially, a printed electrode was fabricated in a paper substrate using carbon nanotube ink, graphite pencil and silver nanoparticle ink. For that the electrode was modified with gold nanoparticles and a molecularly imprintedpolymer (MIP) using CA 15-3 as target molecule. The Atomic Force Microscopy (AFM) images exhibited the change in the morphology after each electrode modification. The roughness increasedafter the electropolymerization, and decreased after the extraction procedure. Next, slightly increased again associated to the interaction of CA 15-3 and the imprinted sites. Finally, the Fourier Transform Infrared Spectroscopy (FTIR) results suggested the extraction/rebinding of CA 15-3 in the MIP sensor and also indicated that the NIP sample do not have specific cavities for the CA 15-3. In short, under optimized conditions, the CNE/AuNP is incubated with CA 15-3 (40 U mL-1) for 2 h at 4 °C. Then the electropolymerization was carried out in the potential range of -0.2 to 1.0 V during 20 cycles at scan rate of 50 mV s-1 using a solution containing 15 mM of oPD. After electropolymerization, the sensor was washed with oxalic acid solution for 2 h, leading to the formation of imprinted cavities. The rebinding process was subsequently constructed for 1 h at 4 °C using CA 15-3 solution. The reproducibility and interference studies showed that the sensor can be reproducible and specific for CA 15-3. Then the sensor was applied in determination of CA 15-3 in samples of serum and saliva. The use in serum presented good recovery, but the application in saliva was not satisfactory. Therefore, the sensor CNE/AuNP/MIP could be used in the determination of CA 15-3 in serum samples.

Label-free electrochemical impedance immunosensor based on modified screen-printed gold electrodes for the diagnosis of canine visceral leishmaniasis.

Leishmaniasis is a disease with high impact on public health in many countries. Visceral leishmaniasis (VL) is a vectorial zoonosis, with dogs as primary reservoirs in the domestic environment. VL presents high morbidity, mortality and importance in epidemiology in the American continent. In the present study, the first label-free electrochemical impedance immunosensor using screen-printed electrodes (SPEs) for the detection of anti-Leishmania infantum antibodies was developed. The soluble antigens of L. infantum were immobilized on an SPE by a 3-mercaptopropionic acid monolayer. Electrochemical impedance spectroscopy (EIS) was used for detecting bimolecular interactions occurring at the electrode surface. The addition of real samples consisting of canine and human sera positive and negative for VL presented high sensitivity and selectivity through EIS. Based on the results, a sensitive, specific, rapid and simple immunosensor was developed successfully with potential application for the serological diagnosis of leishmaniasis disease.

Optimization and Application of Electrochemical Transducer for Detection of Specific Oligonucleotide Sequence for Mycobacterium tuberculosis.

In this study, the electropolymerization of 4-hydroxyphenylacetic acid (4-HPA) over graphite electrodes (GE) was optimized, aiming its application as a functionalized electrochemical platform for oligonucleotides immobilization. It was investigated for the number of potential cycles and the scan rate influence on the monomer electropolymerization by using cyclic voltammetry technique. It was observed that the polymeric film showed a redox response in the region of +0.53/+0.38 V and the increase in the number of cycles produces more electroactive platforms because of the better electrode coverage. On the other hand, the decrease of scan rate produces more electroactive platforms because of the occurrence of more organized coupling. Scanning electron microscopy (SEM) images showed that the number of potential cycles influences the coverage and morphology of the electrodeposited polymeric film. However, the images also showed that at different scan rates a more organized material was produced. The influence of these optimized polymerization parameters was evaluated both in the immobilization of specific oligonucleotides and in the detection of hybridization with complementary target. Poly(4-HPA)/GE platform has shown efficient and sensitive for oligonucleotides immobilization, as well as for a hybridization event with the complementary oligonucleotide in all investigated cases. The electrode was modified with 100 cycles at 75 mV/s presented the best responses in function of the amplitude at the monitored peak current values for the Methylene Blue and Ethidium Bromide intercalators. The construction of the genosensor to detect a specific oligonucleotide sequence for the Mycobacterium tuberculosis bacillus confirmed the results regarding the poly(4-HPA)/GE platform efficiency since it showed excellent sensitivity. The limit of detection and the limit of quantification was found to be 0.56 (±0.05) µM and 8.6 (±0.7) µM, respectively operating with very low solution volumes (15 µL of probe and 10 µL target). The biosensor development was possible with optimization of the probe adsorption parameters and target hybridization, which led to an improvement in the decrease of the Methylene Blue (MB) reduction signal from 14% to 34%. In addition, interference studies showed that the genosensor has satisfactory selectivity since the hybridization with a non-specific probe resulted in a signal decrease (46% lower) when compared to the specific target.

Bioelectrode applied to diagnosis of cardiac disease.

This paper describes the assembly of a bioelectrode based on poly(3-aminophenol) and anti-troponin T antibody for recognition of troponin T, which is a specific biomarker for diagnosis of acute myocardial infarction. This disease causes loss of cellular components, allowing the output of molecules such as troponin T. This proteic component acts as biomarker for diagnosis of acute myocardial infarction due to their high sensitivity and specificity. Poly(3-aminophenol) was electrodeposited onto fluorine doped tin oxide (FTO) coated glass and characterized by spectroscopic methods (UV-Visible, fluorescence, infrared), electrochemical methods (cyclic voltammetry and electrochemical impedance spectroscopy) and morphological methods (laser interferometry, field emission scanning electronic microscopy, and atomic force microscopy). UV/Vis analysis indicated that poly(3-aminophenol) presents extension of conjugation, in according with fluorescence studies. Electrochemical studies indicated that poly(3-aminophenol) electrodeposited in FTO is a material with passivating characteristics for anions and capacity of retaining cationic compounds. Laser interferometry showed that poly(3-aminophenol) covers the FTO surface with a thickness off 375 ± 75 nm. Surface images by FE-SEM and AFM have shown a full coverage on the FTO by the polymer film. The incorporation of anti-troponin T antibody on FTO electrode modified with poly(3-aminophenol) allowed effective and selective detection of cardiac biomarker troponin T, by electrochemical impedance spectroscopy (label free) and by photoluminescence, based on CdSe/ZnS quantum dots. This research shows the step by step assembly of the bioelectrode, used for detection of troponin T by impedimetric and fluorescence methods, opening the opportunity for its use in the diagnosis of others diseases.

Biomarkers for serum diagnosis of infectious diseases and their potential application in novel sensor platforms.

Nanotechnological tools and biomarkers for diagnosis and prognosis, as well as strategies for disease control and monitoring populations at higher risk, are continuous worldwide challenges for infectious diseases. Phage display and monoclonal antibody combinatorial libraries are important sources for biomarker discovery and for improved diagnostic strategies. Mimetic peptides were selected against polyclonal antibodies from patients with dengue fever, leprosy, and leishmaniasis as model diseases, and from immunized chickens with total antigens from all three pathogens. Selected single or combined multi-epitope peptide biomarkers were further associated with four different sensor platforms, classified as affinity biosensors, that may be suitable as general protocols for field diagnosis. We have also developed two methods for nanoparticle agglutination assays (a particle gel agglutination test and a magnetic microparticle [MMP]-enzyme-linked immunosorbent assay [ELISA]) and two electrochemical biosensors (impedimetric and amperometric) for DNA and antibody detection. For the agglutination tests, micro- and nanoparticles were coupled with filamentous bacteriophages displaying the selected mimotopes on their surfaces, which has favored the formation of the antigen-antibody or peptide-protein complexes, amplifying the optical detection in ELISA assays or after the chromatographic separation of the microagglutinates. We have also demonstrated a proof-of-concept for the electrochemical biosensors by using electrodes modified with novel functionalized polymers. These electrochemical biosensors have proven to be fast, very sensitive, and specific for the detection of pathogen DNA and circulating antibodies of patients, which may become important in a wide range of diagnostic devices for many infectious agents.