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MOF-based Electrochemical Sensors for Protein Detection
Published in Ram K. Gupta, Tahir Rasheed, Tuan Anh Nguyen, Muhammad Bilal, Metal-Organic Frameworks-Based Hybrid Materials for Environmental Sensing and Monitoring, 2022
Yang Liu, Juanhua Zhou, Shiyu Zhang, Hongye Wang
At present, many protein detection technologies have been developed, including enzyme-linked immunosorbent assays (ELISA) [3], fluorescence [4], mass spectroscopy (MS) [5], electrochemical methods [2, 6], and so on. The sensitivity of immunofluorescence is very high, but its application in multiple detections is limited due to its inherent shortcomings such as optical background and wide spectral bandwidth [7]. Compared with fluorescence, MS can achieve multiple detections [8]. However, it is difficult in the qualitative and quantitative analysis of low abundance proteins [9]. Moreover, the above two methods are complex, time-consuming, expensive, and require well-trained personnel, which limits their feasibility and portability in practical application [10]. However, the electrochemical method can overcome the above weaknesses. It has high sensitivity, low cost, portability, and simple operation. Therefore, it is widely used in protein analysis of actual samples, especially in the field of point of care and wearable sensing devices [11]. Additionally, combined with appropriate recognition strategies and signal transduction methods, electrochemical sensors based on electrochemical methods have unparalleled advantages in the field of protein sensing.
Microarray 3D Bioprinting for Creating Miniature Human Tissue Replicas for Predictive Compound Screening
Published in Hyun Jung Kim, Biomimetic Microengineering, 2020
Alexander D. Roth, Stephen Hong, Moo-Yeal Lee
While chemical dye stains can characterize specific organelles or quantify general behavior, they are generally toxic to cell health and are quite susceptible to photobleaching due to overexposure and membrane leakage over time. Applications in immunofluorescence allow for targeting specific protein markers in the cell. Because antibodies bind strongly to their target proteins, this method is widely used to detect the upregulation of disease targets. The disadvantage to using immunofluorescence is that antibodies are less likely to penetrate the cell membrane without appropriate permeabilization. Although antibodies bind strongly to their targeted antigen sites, there are occasionally issues with antibodies where binding is non-specific, leading to the detection of false positives. In addition, immunofluorescent stains require aldehyde use during the fixation steps, which cause problems with toxicity. Immunofluorescence has been successfully demonstrated on the microarray chip platforms with cells overexpressing protein markers (Figure 13.5; Kwon et al. 2014; Fernandes et al. 2008). For example, the co-expression levels of three drug-metabolizing enzymes (CYP3A4, CYP2C9, and UGT1A4) expressed in THLE-2 cells on the micropillar chip were assessed using fluorescently labeled antibodies (Kwon et al. 2014; Gustafsson et al. 2014).
Introduction to Biological Light Microscopy
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Jay L. Nadeau, Michael W. Davidson
One of the most long-standing applications of fluorescent dyes is to the technique of immunofluorescence, where a dye-conjugated antibody binds a cell target, showing the distribution of that antigen. Indirect immunofluorescence uses two sets of antibodies: a primary antibody against the antigen of interest, and a secondary dye-coupled antibody that recognizes the primary (Figure 7.29a). The secondary antibodies are according to species: that is, if the primary is made in mouse, the secondary may be made in goat and designated “goat antimouse.” This minimizes the number of fluorescent antibodies that need to be made, ensuring that a commercial fluorescent secondary is always available for any application. Immunofluorescence is nearly always performed on cells that have been fixed and permeabilized with detergents to permit entry of the antibodies. It is especially useful for determining the distribution of a specific antigen (protein or other) within a cell or tissue, and for confirming the expression of a transfected protein (see Practical Tips 7.5 for a basic indirect immunofluorescence protocol).
Analytical challenges and perspectives of assessing viability of Giardia muris cysts and Cryptosporidium parvum oocysts by live/dead simultaneous staining
Published in Environmental Technology, 2022
Kamila Jessie Sammarro Silva, Lyda Patricia Sabogal-Paz
Neither methods B or C, in which Merifluor® reagents were not included, allowed visualizing viable organisms, expected to present green fluorescence under FITC-filter due to esterase activity. Under DAPI-filter (Method B), some G. muris cysts were detected because of their stained nuclei and they were either considered non-viable (if presenting red stain uptake under PI-filter) or they were not visualized showing green fluorescence. An example of this effect is indicated by an arrows in Figure 3, which displays a considered non-viable G. muris cysts, that did not present any green fluorescence under FITC (Figure 3, panel a), but stained brightly in red under PI-filter (Figure 3b). Nevertheless, (oo)cyst identification and detection should not rely on DAPI filter, as Method 1623.1 [24] recommends it only for morphology confirmation after immunofluorescence specific visualization, which is antibody-based.
Mitochondrial uncoupling protein 2 is regulated through heterogeneous nuclear ribonucleoprotein K in lead exposure models
Published in Journal of Environmental Science and Health, Part C, 2020
Gaochun Zhu, Qian Zhu, Wei Zhang, Chen Hui, Yuwen Li, Meiyuan Yang, Shimin Pang, Yaobing Li, Guoyong Xue, Hongping Chen
Antibodies and reagents were obtained commercially as indicated below, including lead acetate (PbAc) (Tianjin benchmark Chemical Reagent Co., Ltd), rabbit anti-UCP2 polyclonal antibody (Bioss company), rabbit anti-hnRNP K polyclonal antibody (Abcam company), mouse anti-β-actin polyclonal antibody (Sigma company), goat anti-rabbit and goat anti-mouse secondary HRP-conjugated antibody (Sigma company), biotin goat anti-rabbit IgG (Boster company), streptavidin-peroxidase (Boster company), TRITC labeled goat anti-rabbit IgG (ImmunoResearch Laboratories Jacksonand), and lipofectamine 2000 (Genepharma company). Rabbit anti-UCP2 polyclonal antibody was used at 1:500 for western blotting, and 1:100 for immunohistochemistry and immunofluorescence. Rabbit anti-hnRNP K polyclonal antibody was used at 1:1000 for western blotting, and 1:500 for immunohistochemistry and immunofluorescence. And mouse anti-β-actin polyclonal antibody was used at 1:1000 for western blotting. Vector and GFP-hnRNP K were kind gifts from Professor Gao XueJuan (from Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, Jinan University, Guangzhou, P. R. China.).
Generation and evaluation of anti-mouse IgG IgY as secondary antibody
Published in Preparative Biochemistry & Biotechnology, 2020
Qi Zhang, Dongyang He, Long Xu, Shikun Ge, Jinquan Wang, Xiaoying Zhang
The production and extraction of IgY has increasingly attracted the interests of the scientific community. It was proved that the availability of IgY as secondary antibody aids in cell level detection and in binding it with different subtypes of IgG.[14] The binding ability of IgY-HRP was verified by random use of different types of mouse IgG antibodies available in the laboratory, and they all showed good binding activity (Fig. 6). Our study on western blot and immunofluorescence demonstrated the ability of IgY as secondary antibody (Figs. 8 and 9), and showed that it is generally comparable to conventional secondary antibody (Fig. 7). There are numerous factors that affect the stability of biological products, thereby influence the experimental results, the most common ones are pH value and temperature. Our study verified the applicability of IgY secondary antibody in complex detection environment (Figs. 10 and 11) with the confirmed potential widely used in complex immunoassay environments.