RESUMEN
In this work, two human epidermal growth factor receptors, HER-1 and HER-2, were selected as biomarkers to enable the detection of breast cancer. Therefore, two biosensors were developed using gold sensor chips coupled with amperometric detection of the enzyme label horse radish peroxidase (HRP). The biosensors/immunosensors relied on indirect sandwich enzyme-linked immunosorbent assays with monoclonal antibodies (Ab) against HER-1 and HER-2 attached to the sensors to capture the biomarkers. Detection polyclonal antibodies followed by secondary anti-rabbit (for HER-1) and anti-goat (for HER-2) IgG antibody-HRP were then applied for signal generation. In buffer, the developed sensors showed limits of detections (LOD) of 1.06 ng mL-1 and 0.95 ng mL-1 and limits of quantification (LOQ) of 2.1 ng mL-1 and 1.5 ng mL-1 for HER-1 and HER-2, respectively. In 100% (undiluted) serum, LODs of 1.2 ng mL-1 and 1.47 ng mL-1 and LOQs of 1.5 ng mL-1 and 2.1 ng mL-1 were obtained for HER-1 and HER-2, respectively. Such limits of detections are within the serum clinical range for the two biomarkers. Furthermore, gold nanoparticles (AuNP) labelled with secondary anti-rabbit and anti-goat IgG antibody-HRP were then used to enhance the assay signal and increase the sensitivity. In buffers, LODs of 30 pg mL-1 were seen for both sensors and LOQs of 98 pg mL-1 and 35 pg mL-1 were recorded for HER-1 and HER-2, respectively. For HER-2 the AuNPs biosensor was also tested in 100% serum obtaining a LOD of 50 pg mL-1 and a LOQ of 80 pg mL-1. The HER-2 AuNP electrochemical immunosensor showed high specificity with very low cross-reactivity to HER-1. These findings demonstrate that the two developed sensors can enable early detection as well as monitoring of disease progression with a beneficial impact on patient survival and clinical outcomes.
Asunto(s)
Técnicas Biosensibles , Neoplasias de la Mama , Nanopartículas del Metal , Humanos , Animales , Conejos , Femenino , Oro , Inmunoensayo , Neoplasias de la Mama/diagnóstico , Inmunoglobulina G , Técnicas ElectroquímicasRESUMEN
Periodontitis is a chronic disease which affects at least 10% of the population. If untreated, periodontitis can lead to teeth loss. Unfortunately, current diagnostic tests are limited in their sensitivity and specificity. In this study, a novel multiplex hand-held colorimetric diagnostic biosensor, using two typical inflammatory salivary biomarkers, Human Neutrophil Elastase (HNE) and Cathepsin-G, was constructed as proof of concept to potentially detect periodontitis. The biosensing method was based on the measurement of proteolytic activity using specific proteases probes. These probes consist of specific proteases substrates covalently bound to a magnetic bead from one end and to the gold sensor surface by the other end. When intact, this renders the golden sensor black. Upon proteolysis, the cleaved magnetic beads will be attracted by an external magnet revealing the golden color of the sensor surface observable by the naked eye. The biosensor was capable of specific and quantitative detection of HNE and Cathepsin-G in solution and in spiked saliva samples with a lower detection limit of 1 pg/mL and 100 fg/mL for HNE and Cathepsin-G, respectively. Examination of periodontitis patients' sample and a healthy control showed the potential of the multiplex biosensor to detect the presence of HNE and Cathepsin-G activity in situ. This approach is anticipated to be a useful biochip array amenable to low-cost point-of-care devices.
Asunto(s)
Técnicas Biosensibles , Catepsina G/análisis , Colorimetría/métodos , Elastasa de Leucocito/análisis , Nanopartículas de Magnetita/química , Periodontitis/diagnóstico , Periodontitis/metabolismo , Biomarcadores/análisis , Catepsina G/metabolismo , Humanos , Elastasa de Leucocito/metabolismoRESUMEN
Glioblastoma multiforme (GBM) is notoriously resistant to treatment. Therefore, new treatment strategies are urgently needed. ATM elicits the DNA damage response (DDR), which confers cellular radioresistance; thus, targeting the DDR with an ATM inhibitior (ATMi) is very attractive. Herein, we show that dynamic ATM kinase inhibition in the nanomolar range results in potent radiosensitization of human glioma cells, inhibits growth and does not conflict with temozolomide (TMZ) treatment. The second generation ATMi analog KU-60019 provided quick, reversible and complete inhibition of the DDR at sub-micromolar concentrations in human glioblastoma cells. KU-60019 inhibited the phosphorylation of the major DNA damage effectors p53, H2AX and KAP1 as well as AKT. Colony-forming radiosurvival showed that continuous exposure to nanomolar concentrations of KU-60019 effectively radiosensitized glioblastoma cell lines. When cells were co-treated with KU-60019 and TMZ, a slight increase in radiation-induced cell killing was noted, although TMZ alone was unable to radiosensitize these cells. In addition, without radiation, KU-60019 with or without TMZ reduced glioma cell growth but had no significant effect on the survival of human embryonic stem cell (hESC)-derived astrocytes. Altogether, transient inhibition of the ATM kinase provides a promising strategy for radiosensitizing GBM in combination with standard treatment. In addition, without radiation, KU-60019 limits growth of glioma cells in co-culture with human astrocytes that seem unaffected by the same treatment. Thus, inter-fraction growth inhibition could perhaps be achieved in vivo with minor adverse effects to the brain.