An immunosensor was developed using a SAM of an alkanethiol associated with PAMAM(G4) dendrimers based on surface plasmon resonance (SPR) to enhance the sensitivity for troponin T detection in blood ...samples. The feasibility of using three-dimensional platforms based on dendrimers for the development of immunosensors was demonstrated by evaluating three different generations of these dendrimers (G3, G4, and G5) to detect troponin T. The results showed the efficiency of these 3D platforms in anchoring biomolecules, amplifying the detection of troponin T. The sandwich assay showed good performance for troponin T detection, using secondary monoclonal antibodies, in the concentration range of 5 – 300 ng mL−1 (0.14 – 8.67 nmol L−1), R2 = 0.991, with the LOD of 3.6 ng mL−1. The sandwich assay’s applicability was demonstrated by evaluating a secondary polyclonal antibody’s performance in the concentration range of 3 – 30 ng mL−1, R2 = 0.998, with the LOD of 0.98 ng mL−1. The immunosensor was applied to determine troponin T in blood plasma samples from healthy patients, with an average recovery of 88 to 104%. The performance of the SPR-based immunosensor indicates reliable results and is expected to contribute to the rapid diagnosis of heart attack, with reduced costs.
The phenomenon of surface plasmon resonance (SPR) through optical sensors was developed from initial studies involving excitation of surface plasmons on metallic substrates. From the beginning, these ...optical systems have attracted increasing interest for application in different areas, ranging from physics, chemistry, and materials science to biology. Although numerous applications have been explored, the use of SPR in the development of biosensors is by far the most prominent. This review provides a brief account of fundamental aspects related to the recent applications of SPR as a tool for the development of new clinical diagnosis methods. The applications of SPR biosensors were illustrated through recent studies published in the field of neglected tropical diseases, with an emphasis on the contributions achieved in visceral leishmaniasis. It was possible to demonstrate the real benefits and the difficulties that the SPR biosensors have encountered in this important and complex system. Finally, future trends in the use of nanomaterials for the development of SPR-based portable devices for application to neglected tropical diseases have been demonstrated.
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•The SPR technique has been used for diagnostic purposes and for the elucidation of biochemical events.•Viability of SPR-based biosensor for the development of new clinical diagnosis methods.•SPR immunosensors showed higher sensitivity for the detection of leishmaniasis antibodies than the ELISA assay.•. The binding affinity values demonstrated high viability of the use of recombinant proteins in control programs.•Perspectives in using nanomaterials and portable device in SPR-based biosensors..
In this study, polypyrrole nanotubes (PPy-NT) and gold nanoparticles (AuNPs) were electrochemically synthesized to form a hybrid material and used as an electroactive layer for the attachment of ...proteins for the construction of a high-performance biosensor. Besides the enhancement of intrinsic conductivity of the PPy-NT, the AuNPs act as an anchor group for the formation of self-assembly monolayers (SAMs) from the gold–sulfur covalent interaction between gold and Mercaptopropionic acid (MPA). This material was used to evaluate the viability and performance of the platform developed for biosensing, and three different biological approaches were tested: first, the Avidin-HRP/Biotin couple and characterizations were made by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), wherein we detected Biotin in a linear range of 100–900 fmol L−1. The studies continued with folate group biomolecules, using the folate receptor α (FR-α) as a bioreceptor. Tests with anti-FR antibody detection were performed, and the results obtained indicate a linear range of detection from 0.001 to 6.70 pmol L−1. The same FR-α receptor was used for Folic Acid detection, and the results showed a limit of detection of 0.030 nmol L−1 and a limit of quantification of 90 pmol L−1. The results indicate that the proposed biosensor is sensitive and capable of operating in a range of clinical interests.
A novel lab-made alginate-based hydrogel device was successfully prepared and applied as a sorption material for the solid-phase microextraction of drugs (fluoxetine and its metabolite, ...norfluoxetine) in human plasma, with subsequent determination by high performance liquid chromatography–fluorescence detection (HPLC-FD). When supported in a polypropylene hollow fiber, the alginate was able to extract the analytes and functioned as a restricted access material, excluding >95 % of proteins from the biological matrix. The results indicate the potential use of this phase/device for quantitative drugs extraction from biological matrices at concentrations compatible with those typical in the literature (0.5 μg mL−1), and with satisfactory precision (13.4 % for fluoxetine and 6.2 % for norfluoxetine). Such outcomes, promoted by a simple and inexpensive material, open a new perspective of exploration of hydrogels as the sorption phase in biological matrices, a concept previously unexplored in the literature.
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•Application of alginate-based hydrogel fibers as sorption material.•Alginate hydrogel acts as a restricted access material for plasma protein exclusion.•Allows direct extraction of drugs from human plasma without needing additional steps such as protein precipitation.•Inexpensive, simple, and efficient sorption phase for drug extraction from biological matrices.
Dendronized gold nanoparticles (AuNPs) were synthesized bearing charged peripheral groups. Two novel AB3-type dendrons were synthesized with a thiol group at the focal point followed by their ...attachment to AuNPs. Dendrons were designed to have nine charged peripheral groups (carboxyl or amine), glycol solubilizing, units and one thiol moiety at the focal point. Both dendrons and all intermediates were synthesized in high yields and characterized by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). The amine- and carboxyl-terminated dendrons were used to functionalize gold nanoparticles (AuNPs) previously stabilized with citrate. The nanoparticles’ diameters and their colloidal stability were investigated using dynamic light scattering (DLS). The size and morphology of the dendronized AuNPs were evaluated by scanning electron microscopy (SEM), which revealed individual particles with no aggregation after replacement of citrate by the dendrons, in agreement with the DLS data. The absorption spectroscopy reveals a prominent plasmonic band at 560 nm for all AuNPs. The zeta potential further confirmed the expected charged structures of the dendronized AuNPs. Considering all the physical–chemical properties of the charged dendronized AuNPs developed in this work, these AuNPs might be used as a weapon against multi-drug resistant bacterial infections.
In this work, a surface plasmon resonance (SPR) immunosensor was developed using an 11-mercaptoundecanoic acid (11-MUA) modified gold SPR sensor chip for the detection of anti-Leishmania infantum ...antibodies. The soluble antigens of L. infantum were securely immobilized on an SPR gold disk by an 11-MUA self-assembled monolayer. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electrochemical microscopy (SECM) techniques were employed in the characterization of the antigen immobilization. After the immunosensor construction, canine serum positive for visceral leishmaniasis was added to its surface and showed significant variation in the SPR angle, indicating excellent sensitivity of the technique for antigen–antibody interaction detection. Moreover, the addition of negative serum was accompanied by a smaller response, demonstrating that the immunosensor shows good specificity against anti-L. infantum antibodies. Therefore, this work demonstrates the successful development of an SPR sensor for anti-L. infantum antibodies detection in short time, showing a great perspective as a sensing system of visceral leishmaniasis in endemic regions.
► A new SPR sensor for anti-L. infantum antibodies detection. ► Surface scans with SECM demonstrate the immobilization of L. infantum antigens. ► The sensor showed good response, allowing analysis of interactions in real time. ► The sensor showed a great perspective as a sensing system to VLC in endemic regions.
Electrically active field-effect transistors (FET) based biosensors are of paramount importance in life science applications, as they offer direct, fast, and highly sensitive label-free detection ...capabilities of several biomolecules of specific interest. In this work, we report a detailed investigation on surface functionalization and covalent immobilization of biomarkers using biocompatible ethanolamine and poly(ethylene glycol) derivate coatings, as compared to the conventional approaches using silica monoliths, in order to substantially increase both the sensitivity and molecular selectivity of nanowire-based FET biosensor platforms. Quantitative fluorescence, atomic and Kelvin probe force microscopy allowed detailed investigation of the homogeneity and density of immobilized biomarkers on different biofunctionalized surfaces. Significantly enhanced binding specificity, biomarker density, and target biomolecule capture efficiency were thus achieved for DNA as well as for proteins from pathogens. This optimized functionalization methodology was applied to InP nanowires that due to their low surface recombination rates were used as new active transducers for biosensors. The developed devices provide ultrahigh label-free detection sensitivities ∼1 fM for specific DNA sequences, measured via the net change in device electrical resistance. Similar levels of ultrasensitive detection of ∼6 fM were achieved for a Chagas Disease protein marker (IBMP8-1). The developed InP nanowire biosensor provides thus a qualified tool for detection of the chronic infection stage of this disease, leading to improved diagnosis and control of spread. These methodological developments are expected to substantially enhance the chemical robustness, diagnostic reliability, detection sensitivity, and biomarker selectivity for current and future biosensing devices.
Poly(3,4-ethylenedioxythiophene) nanotubes (PEDOT-NTs) were electrochemically synthesized onto a stainless steel mesh electrode followed by one-pot electrodeposition of gold nanoparticles (AuNPs) at ...the nanotubular surface. The hybrid nanomaterial has shown remarkable electrochemical properties with the diminishment of the charge transfer resistance besides the possibility of further electrode modification by the thiol bonding at the AuNPs. The modified electrodes were deeply characterized by electrochemical techniques, in special by electrochemical impedance spectroscopy, which provides valuable information about the interfacial processes. The morphology of the nanomaterial was characterized by scanning electron microscopy and transmission electron microscopy images. The biosensing properties were evaluated through the avidin/biotin pair. The methodology of construction of the biosensor was further adapted to explore a cancer biomarker detection, the folate binding protein (FBP), which presented high sensitivity with a limit of detection of 4.5 pmol L–1, one of the lowest found in literature. Also, the hybrid material was used in the development of supercapacitors, the inclusion of AuNPs provided a remarkable durability of the PEDOT-NTs modified electrodes presenting long cycling stability (over 3000 cycles) and high energy density, compared with bare PEDOT nanotubes modified electrodes.
In this work, an SPR immunosensor was developed to elucidate the reaction kinetics between a protein of unknown function in Leishmania infantum (hypothetical C1 protein) and specific antibodies of ...the visceral leishmaniasis (VL). A platform, which is based on layer-by-layer assembly was formed by cysteamine in combination with a fourth-generation poly(amidoamine) dendrimer (PAMAM(G4)) on gold surface for the immobilisation of the protein. This film resulted in amplification of the signal of SPR. Then, a kinetic model based on a bivalent ligation suggested that the reaction between the C1 protein and the anti-C1 antibody occurs in two steps. The value of the equilibrium dissociation constant (KD1×KD2=1.64×10−7molL−1) demonstrated high binding affinity between the biomolecules. Furthermore, low limits of detection (LOD=7.37nmolL−1) and quantification (LOQ=7.83nmolL−1) were presented with the proposed SPR immunosensor. Afterwards, the addition of real samples consisting of positive and negative canine sera for VL was accompanied by high sensitivity and selectivity by SPR immunosensor. Therefore, this study quantitatively demonstrated the strong antigenic character of a hypothetical protein and consequently its potential use in the immunodiagnosis of the VL.
•By the SPR technique, the antigenic character of a hypothetical protein in L. infantum protozoan was kinetically demonstrated.•The dendrimer provided a greater accessible area for interaction with the recombinant C1 antigen.•A proposed kinetic model suggested that the reaction between the C1 antigen and the IgG anti-C1 occurs in two steps.•The SPR immunosensor presented high sensitivity and specificity towards real samples.
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•SbCHIP is a non-spherical symmetric dimer in solution.•SbCHIP forms complexes with Hsp70 and Hsp90 from plant and interacts with E2 enzymes UbcH5 and UbcH13.•SbCHIP is able of ...autoubiquitination, but in the presence of substrate SbCHIP ubiquitinates Hsp70, Hsp90 and luciferase.
The co-chaperone CHIP (carboxy terminus of Hsc70 interacting protein) is very important for many cell activities since it regulates the ubiquitination of substrates targeted for proteasomal degradation. However, information on the structure-function relationship of CHIP from plants and how it interacts and ubiquitinates other plant chaperones is still needed. For that, the CHIP ortholog from Sorghum bicolor (SbCHIP) was identified and studied in detail. SbCHIP was purified and produced folded and pure, being capable of keeping its structural conformation up to 42 °C, indicating that cellular function is maintained even in a hot environment. Also, SbCHIP was able to bind plant Hsp70 and Hsp90 with high affinity and interact with E2 enzymes, performing E3 ligase activity. The data allowed to reveal the pattern of plant Hsp70 and Hsp90 ubiquitination and described which plant E2 enzymes are likely involved in SbCHIP-mediated ubiquitination. Aditionally, we obtained information on the SbCHIP conformation, showing that it is a non-globular symmetric dimer and allowing to put forward a model for the interaction of SbCHIP with chaperones and E2 enzymes that suggests a mechanism of ubiquitination. Altogether, the results presented here are useful additions to the study of protein folding and degradation in plants.
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