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    • Because commercially available immunoassay presents limited

    2018-10-30

    Because commercially available immunoassay presents limited detection sensitivity on detecting low-abundance cancer biomarkers against the background of high-abundance beta amyloid proteins. Recently, gold nanomaterials (GNPs) show unique optical properties and high surface areas, which have been used to improve LOD of biosensors [23–29]. GNPs not only can produce localized field which is significantly amplified through localized surface plasmons (LSPs) excitation, but also can generate a larger quantum yield of fluorescence emission due to increasing the radiative decay rate of fluorophore over 1000-fold, theoretically [30]. These cause the emission of a fluorophore near a GNP being much more enhanced than that in free-space condition; the phenomenon is called metal-enhanced fluorescence (MEF) [31]. Based on the optical properties, we previously proposed and developed an MEF biosensor in order to study protein–protein interactions at ultralow concentration in the range of pg/mL level [4,32–39]. Moreover, the mechanism of fluorescence enhancement using metallic nanoparticles in this biosensor was studied too based on scattering theory [40]. In this research, we use the proposed MEF biosensor to measure HER2 ECD levels and t-PSA levels in human serum samples. To combine HER2 ECD and t-PSA serum levels for diagnostic BC with different subtypes was discussed.
    Material and methods
    Data analysis
    Results and discussion
    Conclusions In this study, we detected the protein levels of HER2 ECD and t-PSA in the serum samples simultaneously utilizing our previously developed MEF biosensor. The significant association between the HER2 status of tumor tissue and the raised HER2 ECD serum level were identified in this study. Our experimental results indicate that the HER ECD serum level could be used for the discrimination between HER2+ and HER2− groups. Simultaneously, our preliminary data also shows that the measuring of serum t-PSA levels is possible for the discrimination between the preoperative BC patients (including HER2+ and HER2− BC subtypes) and the healthy group, because the empirical and fitting AUC are both greater than 0.7. In addition, our preliminary results show that simultaneous monitoring HER2 ECD and t-PSA serum levels exhibits higher AUCs for distinguishing HER2+ BC subtype from HER2− and healthy control groups together (DF-1, AUC>0.9) and also able to differentiate HER2− BC subtype from HER2+ and healthy control groups together (DF-2, AUC>0.8). These results indicate that BC subtypes relates to the specific serum biomarkers of BC and this preliminary study potentially can be introduced a direction on BC diagnosis based on association of HER2-ECD and t-PSA serum levels as a screening biomarker panel for BC.
    Conflict of interest
    Acknowledgments This research was supported by National Science Council of Taiwan (NSC98-2221-E-182-064-MY3). The Chang Gung Memorial Hospital research Grants (CMRPD290091, CMRPD290092 and CMRPD2B0051) are also appreciated.
    Introduction Botulinum neurotoxins are zinc-dependant endopeptidases known to selectively cleave the soluble N-ethylmaleimide sensitive attachment protein receptor (SNARE) group of proteins [2]. Until recently the only approved method of determining the activity of a botulinum neurotoxin sample was the mouse bioassay. This assay involves injected mice intraperitoneally with a sample of the toxin and observing them for symptoms of the disease such as a wasp-waist and paralysis over 4days [16]. The mouse bioassay is capable of detecting 10pg/ml of active toxin; however, it is very lengthy, expensive and has ethical problems due to the use of live animals [14]. In 2011 a cell-based assay developed by Allergan Inc., was granted approval by the FDA to replace the mouse bioassay for the potency testing of BOTOX® [10]. In brief this assay uses neuronal cells on a 96 well plate to which the toxin sample is added. After incubating with the sample the cells are lysed and ELISA performed using antibodies for whole and cleaved SNAP-25. This assay is reported to equal the sensitivity of the mouse bioassay but is still lengthy taking 3days to perform [4]. The major benefit of the mouse bioassay and this cell based assay is the ability to assess the toxins full activity from cell uptake through to proteolytic activity which is vital for the pharmaceutical industry. The development of an assay which can detect and quantify the presence of the toxin at similar or lower limits of detection in a shorter period of time is desirable to improve on the existing methods whilst eliminating the need for live animals. A point of care sensor for suspected botulinum poisoning cases would not need to assess all modes of action just the presence of toxin in a blood sample. This study reports the development of a biosensor for the detection of active botulinum neurotoxin A using gold electrodes modified with the SNARE protein SNAP-25.