Microfluid biosensor for detection of hpv in patient scraping samples: Determining E6 and E7 oncogenes.

E6 and E7 oncogenes are pivotal in the carcinogenic transformation in HPV infections and efficient diagnostic methods can ensure the detection and differentiation of HPV genotype. This study describes the development and validation of an electrochemical, label-free genosensor coupled with a microfluidic system for detecting the E6 and E7 oncogenes in cervical scraping samples. The nanostructuring employed was based on a cysteine and graphene quantum dots layer that provides functional groups, surface area, and interesting electrochemical properties. Biorecognition tests with cervical scraping samples showed differentiation in the voltammetric response. Low-risk HPV exhibited a lower biorecognition response, reflected in ΔI% values of 82.33 % ± 0.29 for HPV06 and 80.65 % ± 0.68 for HPV11 at a dilution of 1:100. Meanwhile, high-risk, HPV16 and HPV18, demonstrated ΔI% values of 96.65 % ± 1.27 and 93 % ± 0.026, respectively, at the same dilution. Therefore, the biorecognition intensity followed the order: HPV16 >HPV18 >HPV06 >HPV11. The limit of detection and the limit of quantification of E6E7 microfluidic LOC-Genosensor was 26 fM, and 79.6 fM. Consequently, the E6E7 biosensor is a valuable alternative for clinical HPV diagnosis, capable of detecting the potential for oncogenic progression even in the early stages of infection.

New bioelectrode based on graphene quantum dots-polypyrrole film and Concanavalin A for pathogenic microorganism detection.

Infections caused by microorganisms are a public health problem worldwide. New biodetection systems are essential to diagnose with accuracy resulting in more effective treatment. In this work, we propose a ConA-conjugated graphene quantum dots and polypyrrole film-based biosensor for label-free detection of Candida albicans, Candida glabrata, Candida tropicalis, E. coli, B. subitilis, and S. aureus. We modified polypyrrole and graphene quantum dots (PPY-QDGs) with Concanavalin A (Con A) lectin. ConA is a glucose/mannose-specific lectin. The results showed that ConA lectin has the highest binding affinity for C. tropicalis and S. subtilis. PPY-GQDs-ConA binding profile revealed differential response for Candida spp (C. tropicalis > C. albicans > C. glabrata) and bacterial (B. subtilis > S. aureus > E. coli). The limits of detection (LOD) obtained were 1.42 CFU/mL for C. albicans, and 3.72 CFU/mL for C. glabrata. C. tropicalis yielded a LOD of 0.18 CFU/mL. The respective LODs for the evaluated bacteria were 0.39 CFU/mL for S. aureus, 0.72 CFU/mL for S. subtilis, and 2.63 CFU/mL for E. coli. The differential response obtained for the sensor can be attributed to the heterogeneous distribution of carbohydrates on the microorganism's surfaces. The proposed system based on a flexible substrate is effective for microbiological diagnosis.

Flexible genosensors based on polypyrrole and graphene quantum dots for PML/RARα fusion gene detection: A study of acute promyelocytic leukemia in children.

Acute promyelocytic leukemia (APL) in children is associated with a favorable initial prognosis. However, minimal residual disease (MRD) follow-up remains poorly defined, and relapse cases are concerning due to their recurrent nature. Thus, we report two electrochemical flexible genosensors based on polypyrrole (PPy) and graphene quantum dots (GQDs) for label-free PML-RARα oncogene detection. Atomic force microscopy (AFM), scanning electron microscope (SEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were used to characterize the technological biosensor development. M7 and APLB oligonucleotide sequences were used as bioreceptors to detect oncogenic segments on chromosomes 15 and 17, respectively. AFM characterization revealed heterogeneous topographical surfaces with maximum height peaks for sensor layers when tested with positive patient samples. APLB/Genosensor exhibited a percentage change in anode peak current (ΔI) of 423 %. M7/Genosensor exhibited a ΔI of 61.44 % for more concentrated cDNA samples. The described behavior is associated with the biospecific recognition of the proposed biosensors. Limits of detection (LOD) of 0.214 pM and 0.677 pM were obtained for APLB/Genosensor and M7/Genosensor, respectively. The limits of quantification (LOQ) of 0.648 pM and 2.05 pM were estimated for APLB/Genosensor and M7/Genosensor, respectively. The genosensors showed reproducibility with a relative standard deviation of 7.12 % for APLB and 1.18 % for M7 and high repeatability (9.89 % for APLB and 1.51 % for M7). In addition, genetic tools could identify the PML-RARα oncogene in purified samples, plasmids, and clinical specimens from pediatric patients diagnosed with APL with high bioanalytical performance. Therefore, biosensors represent a valuable alternative for the clinical diagnosis of APL and monitoring of MRD with an impact on public health.

Electrochemical DNA biosensor for chronic myelocytic leukemia based on hybrid nanostructure.

The present research refers to elaborating a new label-free electrochemical biosensor used to detect the BCR/ABL fusion gene. We used a hybrid nanocomposite composed of chitosan and zinc oxide nanoparticles (Chit-ZnONP) immobilized on a polypyrrole (PPy) film. DNA segments were covalently immobilized, allowing biomolecular recognition. Atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were used to evaluate the assembly stages of the biosensor. The biosensor's analytical performance was investigated using recombinant plasmids containing the target oncogene and clinical samples from patients with chronic myeloid leukemia (CML). A limit of detection (LOD) of 1.34 fM, limit of quantification (LOQ) of 4.08 fM, and sensitivity of 34.03 µA fM-1 cm2 were calculated for the BCR/ABL fusion oncogene. The sensing system exhibited high specificity, selectivity, and reproducibility with a standard deviation (SD) of 4.21%. Additionally, a linear response range was observed between 138.80 aM to 13.88 pM with a regression coefficient of 0.96. Also, the biosensor shows easy operationalization and fast analytical response, contributing to the early cancer diagnosis. The proposed nanostructured device is an alternative for the genetic identification BCR/ABL fusion gene.

Flexible sensor based on conducting polymer and gold nanoparticles for electrochemical screening of HPV families in cervical specimens.

Considering the low sensitivity of cytological exams and high costs of the molecular methods, the development of diagnostic tests for effective diagnosis of HPV infections is a priority. In this work, biosensor composed of polypyrrole (PPy) films and gold nanoparticles (AuNPs) was obtained for specific detection of HPV genotypes. The biosensor was developed by using flexible electrodes based on polyethylene terephthalate (PET) strips coated with indium tin oxide (ITO). Polymeric films and AuNPs were obtained by electrosynthesis. Oligonucleotides sequences modified with functional amino groups were designed to recognize HPV gene families strictly. The modified oligonucleotides were chemically immobilized on the nanostructured platform. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used for the analysis of the electrode modification and monitoring of molecular hybridization. Electrochemical changes were observed after exposure of the biosensors to plasmid samples and cervical specimens. The biosensor based on the BSH16 probe showed a linear concentration range for target HPV16 gene detection of 100 pg µL-1 to 1 fg µL-1. A limit of detection (LOD) of 0.89 pg µL-1 and limit of quantification (LOQ) of 2.70 pg µL-1 were obtained, with a regression coefficient of 0.98. Screening tests on cervical specimens were performed to evaluate the sensibility and specificity for HPV and its viral family. The expression of a biomarker for tumorigenesis (p53 gene) was also monitored. In this work, a flexible system has been successfully developed for label-free detection of HPV families and p53 gene monitoring with high specificity, selectivity, and sensitivity.

Metal-polymer hybrid nanomaterial for impedimetric detection of human papillomavirus in cervical specimens.

The human papillomavirus (HPV) is one of the main sexually transmitted pathogens that infect the anogenital epithelium and mucous membranes. HPV genotypes can be classified as high and low oncogenic risk, with infection by the former resulting in cervical cancer in approximately 100 % of the cases. In this work, we developed an ultrasensitive electrochemical biosensor for the detection and identification of different HPV genotypes. A nanostructured platform based on a matrix of polyaniline (PANI) containing gold nanoparticles (AuNps) was designed for the chemical immobilization of a DNA probe capable of recognizing different HPV types. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM) were used to characterize the genosensor. The impedimetric responses indicate that the proposed sensor was able to detect HPV (types 6, 11, 16, 31, 33, 45, and 58) in cervical specimens (cDNA samples). We obtained different profiles of electrochemical responses for the high and low-risk HPV genotypes. By adopting a three-dimensional quantitative analysis of impedance response variables, it was possible to identify the existence of a pattern of association for samples of high oncogenic risk, which may lead to the differential diagnosis of HPV. The biosensor demonstrated an excellent analytical performance for the detection of HPV genotypes with high sensibility and selectivity. The genosensor exhibited a linear range of response in the 1 pg µL-1 to 100 pg µL-1 range. Besides, a limit of detection (LOD) of 2.74 pg µL-1 and 7.43 pg µL-1 was obtained for HPV11 and HPV16, respectively, with regression coefficients of 99.88 % and 99.47 %. Thus, the proposed sensor may serve as a good prognostic indicator for patients infected with papillomavirus.