A Molecularly Imprinted Polypyrrole/GO@Fe3O4 Nanocomposite Modified Impedimetric Sensor for the Routine Monitoring of Lysozyme.

Lysozyme (LYS) applications encompass anti-bacterial activity, analgesic, and anti-inflammatory effects. In this work, a porous framework that was based on the polymerization of pyrrole (PPy) in the presence of multi-functional graphene oxide/iron oxide composite (GO@Fe3O4) has been developed. Oxygen-containing and amine groups that were present in the nanocomposite were availed to assembly LYS as the molecularly imprinted polymer (MIP) template. The synthesized material (MIPPy/GO@Fe3O4) was electrodeposited on top of a gold microelectrode array. Transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS) were used to confirm the adequate preparation of GO@Fe3O4, and the characterization of the resulting molecularly imprinted electrochemical sensor (MIECS) was carried out by electrochemical impedance spectrometry (EIS), FT-IR analysis, and scanning electron microscopy (SEM). The impedimetric responses were analyzed mathematically by fitting to a Q(Q(RW)) equivalent circuit and quantitative determination of LYS was obtained in a linear range from 1 pg/mL to 0.1 µg/mL, presenting good precision (RSD ≈ 10%, n = 5) and low limit of detection (LOD = 0.009 pg/mL). The fabrication of this device is relatively simple, scalable, rapid, and economical, and the sensor can be used up to nine times without disintegration. The MIECS was successfully applied to the determination of LYS in fresh chicken egg white sample and in a commercial drug, resulting in a straightforward platform for the routine monitoring of LYS.

A systematic review and meta-analysis on the accuracy of rapid immunochromatographic tests for dengue diagnosis.

Rapid immunochromatographic tests are frequently used to diagnose dengue due to their easy use, low cost, and fast response. A high level of accuracy is essential for rapid diagnostic tests to support their large-scale use. Thus, this systematic review aims to evaluate the accuracy of rapid dengue diagnostic tests. The investigation was run through the following databases: LILACS, Medline (Pubmed), CRD, The Cochrane Library, Trip Medical Database, and Google Scholar. To solve difficulties, two independent reviewers performed document screening and selection. ELISA assay was adopted as a reference test because of several methodologic advantages. Seventeen articles were included accordingly, reckoning 6837 participating individuals. The receiver operating characteristic (ROC) and Forest Plot were conducted to evaluate the sensitivity and specificity for each analyzed parameter (anti-dengue IgM, IgG, and NS1 antigen). The risk of bias and quality of evidence were assessed as moderate using QUADAS-2 and Grading of Recommendations Assessment, Development, and Evaluation (GRADE), respectively. The sensitivity of IgM concerning the studied tests ranged from 13.8 to 90%, while that of NS1 ranged from 14.7 to 100% (95% CI). The antibodies with NS1 presented increased sensitivity; pooled data show that the association of the three analytes bestows the best result, with a combined sensitivity of 90% (CI 95%: 87-92%) and a pooled specificity of 89% (CI 95%: 87-92%). Thus, the present review provides relevant knowledge for decision-making between available rapid diagnostic tests.

Electrochemical detection of gram-negative bacteria through mastoparan-capped magnetic nanoparticle.

The increasing number of multidrug resistance microorganisms is an alarming threat, and their rapid detection is essential to prevent nosocomial, foodborne, or waterborne infections. Many peptides derived from the venom of wasp Synoeca surinama have antimicrobial activity against Gram-positive and Gram-negative bacteria. Synoeca-MP, an antimicrobial peptide (AMP) from mastoparan family, seems to increase bacterial membrane permeability, promoting cytotoxicity and membrane disruption. Here Synoeca-MP was evaluated as biorecognition element tethered over chitosan-coated magnetic nanoparticles (Fe3O4-Chit). The transducing layer of the biosensor was developed from the self-assembling of 4-mercaptobenzoic acid (4-MBA) monolayer onto gold substrate. Atomic force microscopy (AFM) analyses confirmed the biointeraction between AMP and different pathogens membranes. The fabrication and performance of the biosensing assembly were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Detection of Enterococcus faecalis (G+), Klebsiella pneumoniae (G-), Pseudomonas aeruginosa (G-), and Candida tropicalis was assessed in a recognition range from 101 to 105 CFU.mL-1. An instrumental limit of detection of 10 CFU.mL-1 was obtained for each specimen. However, the device presented a preferential selectivity towards Gram-negative bacteria. The proposed biosensor is a sensitive, fast, and straightforward platform for microbial detection in aqueous samples, envisaged for environmental monitoring applications.

Electrochemical biosensor based on Temporin-PTA peptide for detection of microorganisms.

Bacterial and fungal infections are challenging due to their low susceptibility and resistance to antimicrobial drugs. For this reason, antimicrobial peptides (AMP) emerge as excellent alternatives to overcome these problems. At the same time, their active insertion into the cell wall of microorganisms can be availed for biorecognition applications in biosensing platforms. Temporin-PTA (T-PTA) is an AMP found in the skin secretions of the Malaysian fire frog Hylarana picturata, which presents antibacterial activity against MRSA, Escherichia coli, and Bacillus subtilis. In this work, T-PTA was explored as an innovative sensing layer aiming for the electrochemical differentiation of Klebsiella pneumoniae, Acinetobacter baumannii, Bacillus subtilis, Enterococcus faecalis, Candida albicans, and C. tropicalis based on the structural differences of their membranes. The biosensor was analyzed through electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). In this approach, the different structural features of each microorganism resulted in different adherence degrees and, therefore, different electrochemical responses. The transducing layer was fabricated by the self-assembling of a 4-mercaptobenzoic acid (MBA) monolayer and gold-capped magnetic nanoparticles (Fe3O4@Au) implemented to improve the electrical signal of the biointeraction. We found that each interaction, expressed in variations of electron transfer resistance and anodic peak current, demonstrated a singular response from which the platform can discriminate all different microorganisms. We found expressive sensitivity towards Gram-negative species, especially K. pneumoniae. A detection limit of 101 CFU.mL-1 and a linear range of 101 to 105 CFU.mL-1 were obtained. The T-PTA biosensor platform is a promising and effective tool for microbial identification.

Self-Enriching Electrospun Biosensors for Impedimetric Sensing of Zika Virus.

Zika virus (ZIKV) infection is associated with the Guillain-Barré syndrome, and when non-vector congenital transmission occurs, fetal brain abnormalities are expected. After ZIKV infection, the blood, breast milk, and other body fluids contain low viral loads. Their detection is challenging as it requires the processing of larger input volumes of the clinical samples. Pre-enrichment is a valuable strategy to increase the analyte concentration. Therefore, the authors propose the use of a hierarchal composite polyaniline-(electrospun nanofiber) hydrogel mat (ENM) for the simultaneous enrichment and impedimetric sensing of ZIKV viral particles. The electrospinning conditions of polyvinyl alcohol and alginate, including blend formulation, were optimized through a factorial design. Disintegration and gelatinization were controlled via cross-linking to improve the hydrogel properties. Hierarchization was achieved by in situ chemical deposition of conductive polyaniline. The carboxyl groups of the ENM were used for the covalent immobilization of anti-ZIKV polyclonal antibodies used in the specific recognition of ZIKV within the medium of Vero cell culture. The specific capture and desorption of virions were studied at different pHs. ENMs were characterized by scanning electron microscopy and FTIR. Atomic force microscopy along with UV-vis and electrochemical impedance spectroscopies was used to monitor the antibody immobilization, ZIKV capture, and elution processes. Our results show that 14.2 mg (0.25 cm3) of ENM can capture 38.7 ± 2.5 µg of ZIKV with a desorption rate of 99.97% (38.29 ± 2.7 µg ZIKV), which is reusable for at least three times. Therefore, the capture capacity (micrograms of ZIKV captured per milligram of ENM) of polyaniline-hierarchized mats was 2.72 µg ZIKV/mg. The impedance LOD value was determined to be 2.76 µg of ZIKV particles (approximately 6.6 × 103 PFU/mL). As a result, we present a fast small-scale purification system that can simultaneously monitor ZIKV electrochemically and optically.

Nanostructured sensor platform based on organic polymer conjugated to metallic nanoparticle for the impedimetric detection of SARS-CoV-2 at various stages of viral infection.

The projection of new biosensing technologies for genetic identification of SARS-COV-2 is essential in the face of a pandemic scenario. For this reason, the current research aims to develop a label-free flexible biodevice applicable to COVID-19. A nanostructured platform made of polypyrrole (PPy) and gold nanoparticles (GNP) was designed for interfacing the electrochemical signal in miniaturized electrodes of tin-doped indium oxide (ITO). Oligonucleotide primer was chemically immobilized on the flexible transducers for the biorecognition of the nucleocapsid protein (N) gene. Methodological protocols based on cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM) were used to characterize the nanotechnological apparatus. The biosensor's electrochemical performance was evaluated using the SARS-CoV-2 genome and biological samples of cDNA from patients infected with retrovirus at various disease stages. It is inferred that the analytical tool was able to distinguish the expression of SARS-CoV-2 in patients diagnosed with COVID-19 in the early, intermediate and late stages. The biosensor exhibited high selectivity by not recognizing the biological target in samples from patients not infected with SARS-CoV-2. The proposed sensor obtained a linear response range estimated from 800 to 4000 copies µL-1 with a regression coefficient of 0.99, and a detection limit of 258.01 copies µL-1. Therefore, the electrochemical biosensor based on flexible electrode technology represents a promising trend for sensitive molecular analysis of etiologic agent with fast and simple operationalization. In addition to early genetic diagnosis, the biomolecular assay may help to monitor the progression of COVID-19 infection in a novel manner.

Concanavalin A differentiates gram-positive bacteria through hierarchized nanostructured transducer.

Biosensors are pre-prepared diagnostic devices composed of at least one biological probe. These devices are envisaged for the practical identification of specific targets of microbiological interest. In recent years, the use of narrow-specific probes such as lectins has been proven to distinguish bacteria and glycoproteins based on their superficial glycomic pattern. For instance, Concanavalin A is a carbohydrate-binding lectin indicated as a narrow-specific biological probe for Gram-negative bacteria. As a drawback, Gram-positive bacteria are frequently overlooked from lectin-based biosensing studies because their identification results in low resolution and overlapped signals. In this work, the authors explore the effect that platform nanostructuration has over the electrochemical response of ConA-based platforms constructed for bacterial detection; one is formed of chitosan-capped magnetic nanoparticles, and another is composed of gold nanoparticle-decorated magnetic nanoparticles. The biosensing platforms were characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) as a function of bacterial concentration. Our results show that probe-target interaction causes variations in the electrical responses of nanostructured transducers. Moreover, the association of gold nanoparticles to magnetic nanoparticles resulted in an electrical enhancement capable of overcoming low resolution and overlapping Gram-positive identification. Both platforms attained a limit of detection of 10 ° CFU mL-1, which is useful for water analyses and sanitation concerns, where low CFU mL-1 are always expected. Although both platforms were able to detect Gram-negative bacteria, Gram-positives were only correctly differentiated by the gold nanoparticle-decorated magnetic nanoparticles, thus demonstrating the positive influence of hierarchically nanostructured platforms.

Nanostructured electrochemical immunosensor for detection of serological alkaline phosphatase.

Alkaline phosphatase (ALP) is an enzyme that plays an important role in bone mineralization and skeletal growth. Variations in physiological levels of ALP have been correlated to diseases such as osteomalacia, Paget's disease and arterial calcifications. In this context, the integration of carbon nanotubes (CNT) within osteointegration implants has shown to increase ALP's mineralization activity in virtue of their surface chemistry and their morphological resemblance to collagen nanofibers. In this study we present the development and analytical application of an impedimetric immunosensor based in gold nanoparticle-decorated CNT, which characteristics are desirable in implantable biosensors. The device effectively detects ALP within blood serum, a complex biological fluid where most expressed proteins can be found. Robustness and high sensitivity were attained by immobilizing covalently anti-ALP antibody as a specific probe towards ALP. Cyclic voltammetry, electrochemical impedance spectroscopy and atomic force microscopy were used to characterize the sensor system throughout mounting steps and real sample testing. Impedimetric responses were adjusted to a theoretical electrical circuit and charge transfer resistance showed to be an adequate parameter to evaluate the biorecognition process of the analyte. Additionally, amperometrical current variation and changes in topography found over the surface after positive samples evidenced biorecognition. The final biosensor showed excellent performance with two linear ranges from 0.5 to 50 IU.L-1 and from 100 to 600 IU.L-1; limits of detection were calculated as 0.25 and 84.6 IU.L-1 respectively with a relative standard deviation lower than 5%. The device was found to be selective, avoiding protein c, a potential interferer occurring during inflammatory processes. The proposed strategy is promising for osteogenic applications where it can improve osteointegration implants by monitoring ALP activity.

Clavanin A-bioconjugated Fe3O4/Silane core-shell nanoparticles for thermal ablation of bacterial biofilms.

The use of central venous catheters (CVC) is highly associated with nosocomial blood infections and its use largely requires a systematic assessment of benefits and risks. Bacterial contamination of these tubes is frequent and may result in development of microbial consortia also known as biofilm. The woven nature of biofilm provides a practical defense against antimicrobial agents, facilitating bacterial dissemination through the patient's body and development of antimicrobial resistance. In this work, the authors describe the modification of CVC tubing by immobilizing Fe3O4-aminosilane core-shell nanoparticles functionalized with antimicrobial peptide clavanin A (clavA) as an antimicrobial prophylactic towards Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. Its anti-biofilm-attachment characteristic relies in clavA natural activity to disrupt the bacterial lipidic membrane. The aminosilane shell prevents iron leaching, which is an important nutrient for bacterial growth. Fe3O4-clavA-modified CVCs showed to decrease Gram-negative bacteria attachment up to 90% when compared to control clean CVC. Additionally, when hyperthermal treatment is triggered for 5 min at 80 °C in a tubing that already presents bacterial biofilm (CVC-BF), the viability of attached bacteria reduces up to 88%, providing an efficient solution to avoid changing catheter.

Real-time monitoring of amyloid fibrillation by electrical impedance spectroscopy.

Electrical impedance spectroscopy (EIS) appears a promising label-free methodology for the investigation of processes related to the aggregation of macromolecules in solution. Here, we explore the EIS technique as a convenient tool for studying the irreversible aggregation of human insulin and describing its corresponding fibrillation kinetics. The in situ measurement of the electrical response of pure insulin solutions at 60°C allows for the real-time monitoring of the protein fibrillation as a function of the incubation time. The fitting of the EIS data through an equivalent circuit based on a constant phase element provides a simple set of electric parameters whose abrupt changes can be associated to transitions occurring in the organization of the macromolecules. For establishing the reliability of the method proposed, we have compared the protein aggregation profile collected from the EIS data to that obtained from a conventional fluorescence methodology where Thioflavin T (ThT) is used as a dye probe. The description of the fibrillation process is quite similar in both cases, since characteristic times of the same order were found for the consecutive processes associated to the initial lag phase of insulin fibrillation, to the rapid growth of amyloidal aggregates and to the final saturation step. Our results suggest that in situ EIS can be considered as a promising approach for the real-time label-free monitoring of protein fibril formation.

Biosensing breast cancer cells based on a three-dimensional TIO2 nanomembrane transducer.

The early diagnosis of breast cancer is crucial for the successful treatment and recovery phases of the patients suffering from the disease. Although mammography is considered the gold standard for diagnosis, it fails to detect some cancers in high-density breasts. In this work, we propose for the first time a tridimensional biosensor platform, to be used on an electrochemical point-of-care device. The bioconjugated platform is constructed on a series of covalent linkages between lectin molecules and a cysteine layer immobilized over gold-coated TiO2 butterfly-like tridimensional nanomembranes. Through the use of vegetal lectins, we managed to take advantage of the markedly atypical glycomic profile of the cancerous mammalian cell membrane and successfully made a distinction between highly invasive (T47D) and less invasive (MCF7) cancer cell lines. The selectivity of the biosensor was tested by using normal human skin-fibroblast. The proposed cytosensor demonstrated limits of detection as low as 10 cells mL-1 for every cell line and a linear range from 10 to 1.0×106 cells mL-1. Considering that electrochemical impedance values can be correlated with the number of breast cancer cells present in the sample, we suggest that the proposed platform could be useful in facilitating the diagnosis of cancer.

Attomolar electrochemical detection of the BCR/ABL fusion gene based on an amplifying self-signal metal nanoparticle-conducting polymer hybrid composite.

In the last ten years, conjugated polymers started to be used in the immobilization of nucleic acids via non-covalent interactions. In the present study, we describe the construction and use of an electrochemical DNA biosensor based on a nanostructured polyaniline-gold composite, specifically developed for the detection of the BCR/ABL chimeric oncogene. This chromosome translocation is used as a biomarker to confirm the clinical diagnosis of both chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). The working principle of the biosensor rests on measuring the conductivity resulting from the non-covalent interactions between the hybrid nanocomposite and the DNA probe. The nanostructured platform exhibits a large surface area that enhances the conductivity. Positive cases, which result from the hybridization between DNA probe and targeted gene, induce changes in the amperometric current and in the charge transfer resistance (RCT) responses. Atomic force microscopy (AFM) images showed changes in the genosensor surface after exposure to cDNA sample of patient with leukemia, evidencing the hybridization process. This new hybrid sensing-platform displayed high specificity and selectivity, and its detection limit is estimated to be as low as 69.4 aM. The biosensor showed excellent analytical performance for the detection of the BCR/ABL oncogene in clinical samples of patients with leukemia. Hence, this electrochemical sensor appears as a simple and attractive tool for the molecular diagnosis of the BCR/ABL oncogene even in early-stage cases of leukemia and for the monitoring of minimum levels of residual disease.