Electro-immunosensor for ultra-sensitive determination of cardiac troponin I based on reduced graphene oxide and polytyramine.

This work reports the construction of a novel nanostructured immunosensor for detection of the troponin I biomarker (cTnI). Anti-troponin I antibody was anchored on the modified graphite electrode with reduced graphene oxide and polytyramine for detection of troponin I in serum samples. The performance of the electro-immunosensor was evaluated by differential pulse voltammetry. The immunosensor presented a wide work range, from 4 ng mL-1 to 4 pg mL-1 , whose detection limit (4 pg mL-1 ) is significantly lower than the basal level in human serum, and maintained 100% of response after 30 days of storage. Moreover, the immunosensor showed good selectivity for detection of cTnI in real sample containing interfering substances and specificity of response to cTnI in the serum of healthy and sick patients, and demonstrated the possibility of reuse for two consecutive analyses, in addition to using a simplified and inexpensive platform when compared to other devices, demonstrating them excellent potential for application in diagnosis in the early stages of acute myocardial infarction.

Application of nanomaterials for the electrical and optical detection of the hepatitis B virus.

This work describes different approaches for the detection of hepatitis B virus (HBV) genomic DNA, using electrochemical and optical techniques. The platforms consisted of a single-stranded DNA probe (HEPB1S), specific to HBV, grafted on a gold electrode modified with reduced graphene oxide or gold nanoparticles. Differential pulse voltammetry analysis indicates that the addition of HBV genomic DNA caused an increase of about 1.4 times in the current peak value, when compared to the negative control. It was observed a linear dependence with the log HBV-genomic DNA concentration and the electrochemical biosensor detected until 7.65 pg µL-1 of the target. Electrochemical impedance spectroscopy measurements showed an increase of about 2 times in the charge transfer resistance, after the addition of HBV genomic DNA. Assays using colloidal suspension of gold nanoparticles showed a shift of the peak wavelength, linearly proportional to the HBV-genomic DNA concentration, with a detection limit of 0.15 ng µL-1. The applicability of the gold nanoparticles for clinical samples was tested with success in the blood plasma. All the approaches used in this work were effective in detecting genomic DNA or blood plasma in positive samples for HBV.

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.

Detection of a specific biomarker for Epstein-Barr virus using a polymer-based genosensor.

This paper describes methodology for direct and indirect detections of a specific oligonucleotide for Epstein-Barr virus (EBV) using electrochemical techniques. The sequence of oligonucleotide probe (EBV1) revealed a high sequence identity (100%) with the EBV genome. For the development of the genosensor, EBV1 was grafted to the platform sensitized with poly(4-aminothiophenol). After that, the hybridization reaction was carried out with the complementary target (EBV2) on the modified electrode surface using ethidium bromide as DNA intercalator. The oxidation peak currents of ethidium bromide increased linearly with the values of the concentration of the complementary sequences in the range from 3.78 to 756 µmol·L⁻¹. In nonstringent experimental conditions, this genosensor can detect 17.32 nmol·L⁻¹ (three independent experiments) of oligonucleotide target, discriminating between complementary and non-complementary oligonucleotides, as well as differentiating one-base mismatch, as required for detection of genetic diseases caused by point mutations. The biosensor also displayed high specificity to the EBV target with elimination of interference from mix (alanine, glucose, uric acid, ascorbic acid, bovine serum albumin (BSA), glutamate and glycine) and good stability (120 days). In addition, it was possible to observe differences between hybridized and non-hybridized surfaces through atomic force microscopy.

Fentogram electrochemical detection of HIV RNA based on graphene quantum dots and gold nanoparticles.

This work reports the construction of an HIV-specific genosensor through the modification of carbon screen-printed electrodes (CSPE) with graphene quantum dots decorated with L-cysteine and gold nanoparticles (cys-GQDs/AuNps). Cys-GQDs were characterized by FT-IR and UV-vis spectra and electronic properties of the modified electrodes were evaluated by cyclic voltammetry and electrochemical impedance spectroscopy. The modification of the electrode surface with cys-GQDs and AuNps increased the electrochemical performance of the electrode, improving the electron transfer of the anionic redox probe [Fe(CN)6]3-/4- on the electrochemical platform. When compared to the bare surface, the modified electrode showed a 1.7 times increase in effective electrode area and a 29 times decrease in charge transfer resistance. The genosensor response was performed by differential pulse voltammetry, monitoring the current response of the anionic redox probe, confirmed with real genomic RNA samples, making it possible to detect 1 fg/mL. In addition, the genosensor maintained its response for 60 days at room temperature. This new genosensor platform for early detection of HIV, based on the modification of the electrode surface with cys-GQDs and AuNps, discriminates between HIV-negative and positive samples, showing a low detection limit, as well as good specificity and stability, which are relevant properties for commercial application of biosensors.

Advancement in SARS-CoV-2 diagnosis: A new and stable electrochemical biosensor for genomic RNA detection.

Coronavirus disease (COVID-19) is caused by infection with the SARS-CoV-2 virus, having already caused more than seven million deaths worldwide. Conventional techniques for SARS-CoV-2 detection have limitations, as high cost, low specificity, and longer analysis time, among others. Biosensors emerge as a necessary alternative to overcome the difficulties of current diagnostics. This paper reports a sensor platform where silver-doped zinc oxide nanomaterial (Ag:ZnONp) was used onto carbon screen-printed electrode and ethidium bromide as indicator for development of a specific electrochemical genosensor for COVID-19. This genosensor demonstrated good linearity between the concentrations of 5.62 × 104-5.62 copies/mL and a detection limit of 5 copies/mL with gRNA in patient's samples, with a response time within 30 min. Molecular modeling and morphological analysis are in agreement with obtained electrochemical results. Additional techniques such as AFM, SEM, and EIS were conducted to characterize the morphological and electrochemical properties of the biosensor's surface. The biosensor was also capable of detecting the target presence in spiked samples and demonstrated a stability of 60 days, higher than other similar biosensors for SARS-CoV-2.

A highly reusable genosensor for late-life depression diagnosis based on microRNA 184 attomolar detection in human plasma.

Late-Life Depression (LLD) is one of the most prevalent psychiatric disorders in elderly, causing significant functional impairments. MicroRNAs are small molecules involved in the post-transcriptional regulation of gene expression. Elderly individuals diagnosed with LLD present down regulation of miR-184 (hsa-miR-184) expression compared to healthy patients. Therefore, this miR-184 can be used as a biomarker to diagnose LLD. Current LLD diagnosis depends primarily on clinical subjective identification, based on symptoms and variable scales. This work introduces a novel and facile approach for the LLD diagnosis based on the development of an electrochemical genosensor for miR-184 detection in plasma, using differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). DPV results presented a 2-Fold increase in current value for healthy patients, compared to individuals with LLD when monitoring ethidium bromide oxidation peak. For EIS, a 1.5-fold increase in charge transfer resistance for healthy elderly subjects was observed in comparison with depressed patients. In addition, the analytical performance of the biosensor was evaluated using DPV, obtaining a linear response ranging from 10-9 mol L-1 to 10-17 mol L-1 of miR-184 in plasma and a detection limit of 10 atomoles L-1. The biosensor presented reusability, selectivity and stability, the current response remained 72% up to 50 days of storage. Thus, the genosensor proved to be efficient in the diagnosis of LLD, as well as the accurate quantification of miR-184 in real plasma samples of healthy and depressed patients.

Label-free and reagentless electrochemical genosensor based on graphene acid for meat adulteration detection.

With the increased demand for beef in emerging markets, the development of quality-control diagnostics that are fast, cheap and easy to handle is essential. Especially where beef must be free from pork residues, due to religious, cultural or allergic reasons, the availability of such diagnostic tools is crucial. In this work, we report a label-free impedimetric genosensor for the sensitive detection of pork residues in meat, by leveraging the biosensing capabilities of graphene acid - a densely and selectively functionalized graphene derivative. A single stranded DNA probe, specific for the pork mitochondrial genome, was immobilized onto carbon screen-printed electrodes modified with graphene acid. It was demonstrated that graphene acid improved the charge transport properties of the electrode, following a simple and rapid electrode modification and detection protocol. Using non-faradaic electrochemical impedance spectroscopy, which does not require any electrochemical indicators or redox pairs, the detection of pork residues in beef was achieved in less than 45 min (including sample preparation), with a limit of detection of 9% w/w pork content in beef samples. Importantly, the sample did not need to be purified or amplified, and the biosensor retained its performance properties unchanged for at least 4 weeks. This set of features places the present pork DNA sensor among the most attractive for further development and commercialization. Furthermore, it paves the way for the development of sensitive and selective point-of-need sensing devices for label-free, fast, simple and reliable monitoring of meat purity.

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.

DNA electrochemical biosensor for detection of Alicyclobacillus acidoterrestris utilizing Hoechst 33258 as indicator.

Alicyclobacillus acidoterrestris is an acidophilic and thermophilic bacterium present in the soil, often associated with the spoilage of acidic juices, such as orange juice. Their spores resist pasteurization and, when reactivated, modify the organoleptic properties of the juice, making it unsuitable for consumption, due mainly to production of guaiacol. Biosensors are detection devices that respond quickly and are easy to handle, with great potential for use in the juice production chain. In this context, this work reports an electrochemical genosensor for detection of A. acidoterrestris, based on a graphite electrode modified with electrochemically reduced graphene oxide, a polymer derived from 3-hydroxybenzoic acid and a specific DNA probe sequence complementary with the genomic DNA of A. acidoterrestris. Detection of the target was performed by monitoring the oxidation peak of the Hoechst 33258, a common DNA stainer. The genosensor detection limit was 12 ng mL-1 and it kept 77% of response after ten weeks, and a test showed that orange juice does not interfere with bacteria lysate detection. This biosensor is the first platform for electrochemical detection of the genomic DNA of A. acidoterrestris in the literature, and the first to use Hoechst 33258 as indicator with whole genomic DNA molecules.

Ninhydrin as a novel DNA hybridization indicator applied to a highly reusable electrochemical genosensor for Candida auris.

This work reports a simple strategy for Candida auris genomic DNA (gDNA) detection, a multi-resistant fungus associated with nosocomial outbreaks in healthcare settings, presenting high mortality and morbidity rates. The platform was developed using gold electrode sensitized with specific DNA capture probe and ninhydrin as a novel DNA hybridization indicator. The genosensor was able to detect C. auris in urine sample by differential pulse voltammetry and electrochemical impedance spectroscopy. The biosensor's analytical performance was evaluated by differential pulse voltammetry, detecting up to 4.5 pg µL-1 of C. auris gDNA in urine (1:10, V/V). Moreover, the genosensor was reused eight times with no loss in the current signal response. The genosensor showed selectivity and stability, maintaining 100% of its response up to 80 days of storage. In order to analyze interactions of single and double-stranded DNA with ninhydrin, SEM, AFM and molecular dynamics studies followed by docking simulations were performed. Theoretical calculations showed ninhydrin interactions more favorably with dsDNA in an A-T rich binding pocket rather than with the ssDNA. Therefore, the proposed system is a promising electrochemical detection device towards a more accurate detection of C. auris gDNA in biological samples.

A novel peptide-based sensor platform for detection of anti-Toxoplasma gondii immunoglobulins.

Toxoplasma gondii is an intracellular protozoan parasite responsible for toxoplasmosis, which affects humans and animals. Serologic detection of anti-T. gondii immunoglobulins plays a crucial role in the clinical diagnosis of toxoplasmosis. In this work, a novel electrochemical immunosensor for detecting anti-T. gondii immunoglobulins is reported, based on immobilization of an in silico predicted peptide (PepB3), obtained from membrane protein of T. gondii, on the graphite electrode modified with poly(3-hydroxybenzoic acid). Indirect ELISA confirmed infection and binding specificity of peptide PepB3. Molecular modelling and simulations show this peptide binds to the T. gondii human Fab antibody in the surface antigen 1 (SAG1) binding site, remaining a stable complex during the molecular dynamic simulations, especially by hydrogen bonds and hydrophobic interactions. This electrochemical immunosensor was able to discriminate different periods of infection, using infected mouse serum samples, showing selectivity and discriminating infected and uninfected mouse serum.

Electrochemical Investigation of oligonucleotide-DNA hybridization on poly(4-methoxyphenethylamine).

This work describes the immobilization of purine and pyrimidine bases and immobilization/hybridization of synthetic oligonucleotides on graphite electrodes modified with poly(4-methoxyphenethylamine) produced in acid medium. The immobilization of adenine, guanine, cytosine and thymine on these modified electrodes was efficient, producing characteristic peaks. Another relevant observation is that, according to the literature, pyrimidine bases, cytosine and thymine are more difficult to detect. However, when immobilized onto the poly(4-methoxyphenethylamine), a significant increase in the magnitude of the current was obtained. The observation of the hybridization between the poly(GA) probe and its complementary, poly(CT) target, was possible by monitoring the guanosine and adenosine peaks or through methylene blue indicator, using differential pulse voltammetry. Hybridization results in a decrease of the peak current of guanosine and adenosine or the signal of methylene blue accumulated on the modified electrode surface. The hybridization with the complementary target was also investigated by electrochemical impedance spectroscopy. The results showed a significant modification in the Nyquist plot, after addition of the complementary target, with increase of the charge transference resistance.

Development of direct assays for Toxoplasma gondii and its use in genomic DNA sample.

This work describes an approach for the selection and detection of specific DNA probes related to Toxoplasma gondii, a protozoan parasite responsible for toxoplasmosis. The detection system was developed on graphite carbon electrode modified with poly(3-hydroxybenzoic acid) sensitized with ToxG1 probe. The hybridization of the specific genomic DNA related to T. gondii showed good response by direct detection of guanine residue oxidation using differential pulse voltammetry (DPV). The biosensor was able to distinguish both the complementary and non-complementary targets and detect up to 100ngµL-1 of the T. gondii genomic DNA. The hybridization (ToxG1: T. gondii genomic DNA) was confirmed by optical measurement. Optical assays using gold nanoparticles:ToxG1 probe showed a significant change in the absorbance peak in the presence of the T. gondii genomic DNA according to the electrochemical results. This novel biosensor shows potential as electrochemical transducer and was successfully applied in the biological sample.

A novel reactive epitope-based antigen targeted by serum autoantibodies in oligoarticular and polyarticular juvenile idiopathic arthritis and development of an electrochemical biosensor.

Currently, there are no specific markers for juvenile idiopathic arthritis (JIA) diagnosis, which is based on clinical symptoms and some blood tests for diseases' exclusion. Aiming to select new epitope-based antigens (mimotopes) that could recognize circulating autoantibodies in most JIA forms, we screened a phage displayed random peptide library against IgG antibodies purified from serum of JIA patients. ELISA assay was carried out to confirm immunoreactivity of selected peptides against sera IgG antibodies from JIA patients, healthy children and patients with other autoimmune diseases. The mimotope PRF+1 fused to phage particles was able to efficiently discriminate JIA patients from controls, and for this reason was chosen to be chemically synthesized for validation in a larger sample size. The synthetic peptide was immobilized onto bioelectrodes' surface for antibody detection by electrochemical analyses through differential pulse voltammetry. The PRF+1 synthetic peptide has efficiently discriminated JIA patients from control groups (p<0.0001) with a very good accuracy (AUC>0.84; sensitivity=61%; specificity=91%). The electrochemical platform proved to be fast, low cost and effective in detecting anti-PRF+1 antibodies from JIA patients compared to healthy controls (p=0.0049). Our study describes a novel and promising epitope-based biomarker for JIA diagnosis that can become a useful tool for screening tests, which was successfully incorporated onto an electrochemical biosensor and could be promptly used in field diagnostics.

Functional epitope core motif of the Anaplasma marginale major surface protein 1a and its incorporation onto bioelectrodes for antibody detection.

Anaplasmosis, a persistent intraerythrocytic infection of cattle by Anaplasma marginale, causes severe anemia and a higher rate of abortion, resulting in significant loss to both dairy and beef industries. Clinical diagnosis is based on symptoms and confirmatory laboratory tests are required. Currently, all the diagnostic assays have been developed with whole antigens with indirect ELISA based on multiple epitopes. In a pioneer investigation we demonstrated the use of critical motifs of an epitope as biomarkers for immunosensor applications. Mimotopes of the MSP1a protein functional epitope were obtained through Phage Display after three cycles of selection of a 12-mer random peptide library against the neutralizing monoclonal antibody 15D2. Thirty-nine clones were randomly selected, sequenced, translated and aligned with the native sequence. The consensus sequence SxSSQSEASTSSQLGA was obtained, which is located in C-terminal end of the 28-aa repetitive motif of the MSP1a protein, but the alignment and sequences' variation among mimotopes allowed us to map the critical motif STSSxL within the consensus sequence. Based on these results, two peptides were chemically synthesized: one based on the critical motif (STSSQL, Am1) and the other based on the consensus sequence aligned with the native epitope (SEASTSSQLGA, Am2). Sera from 24 infected and 52 healthy animals were tested by ELISA for reactivity against Am1 and Am2, which presented sensitivities of 96% and 100%, respectively. The Am1 peptide was incorporated onto a biolectrode (graphite modified with poly-3-hydroxyphenylacetic acid) and direct serum detection was demonstrated by impedance, differential pulse voltammetry, and atomic force microscopy. The electrochemical sensor system proved to be highly effective in discriminating sera from positive and negative animals. These immunosensors were highly sensitive and selective for positive IgG, contaminants did not affect measurements, and were based on a simple, fast and reproducible electrochemical system.

A single amino acid substitution in one of the lipases of Aspergillus nidulans confers resistance to the antimycotic drug undecanoic acid.

A plausible approach to evaluate the inhibitory action of antifungals is through the investigation of the fungal resistance to these drugs. We describe here the molecular cloning and initial characterization of the A. nidulans lipA gene, where mutation (lipA1) conferred resistance to undecanoic acid, the most fungitoxic fatty acid in the C(7:0)-C(18:0) series. The lipA gene codes for a putative lipase with the sequence consensus GVSIS and WIFGGG as the catalytic signature. Comparison of the wild-type and LIP1 mutant strain nucleotide sequences showed a G --> A change in lipA1 allele, which results in a Glu(214) --> Lys substitution in LipA protein. This ionic charge change in a conserved LipA region, next to its catalytic site, may have altered the catalytic properties of this enzyme resulting in resistance to undecanoic acid.