Detection of biotin with zeptomole sensitivity using recombinant spores and a competition assay
- PDF / 675,006 Bytes
- 8 Pages / 595.276 x 790.866 pts Page_size
- 35 Downloads / 162 Views
RESEARCH PAPER
Detection of biotin with zeptomole sensitivity using recombinant spores and a competition assay Wen-Zhi Lin 1 & Wan-Chun Liao 2 & Fang-Hsi Chen 3 & I-Cheng Ma 3 & Shao-Yi Hou 2 Received: 17 April 2020 / Revised: 22 July 2020 / Accepted: 30 July 2020 / Published online: 5 August 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Detection of protein-binding analytes is important for many applications. Currently, various instrument-based techniques are used for detecting protein-binding analytes. However, such techniques have several limitations including high cost and time-consuming sample processing. In order to overcome these limitations, we developed a sensitive competition assay for the detection of protein-binding analytes using recombinant endospores as a sensing element. The method is based on the competition between the biotin, the model analyte, and a biotin-magnetic bead complex to bind the recombinant spores containing the biotin binding region of streptavidin. After magnetic attraction, the residual spores in the suspension are spread on plates to form colonies which are used to count the amount of the residual spores; the higher the residual ratio of spores, the more biotin in the samples. The linear range was from 150 zmol to 1.5 fmol and the limit of detection of the assay was 150 zmol. The assay proposed herein is sensitive and does not require any expensive equipment. It is suitable for qualitative or semi-quantitative analysis such as screening tests for the detection of toxic chemicals. Keywords Protein-binding analyte detection . Competition assay . Recombinant endospores . Magnetic bead . Biotin . Streptavidin
Introduction In biochemistry, many analytes specifically and strongly bind certain proteins. For example, haptens, such as biotin [1], dioxin [2], marijuana [3], and many other compounds [1], alone rarely cause immune responses but still specifically bind to their antibodies, the specialized immune proteins. Furthermore, biotin tightly binds avidin or streptavidin with a dissociation constant of the order of 10−14 mol/L [4], and this characteristic is often used in
* Shao-Yi Hou [email protected] 1
Institute of Preventive Medicine, National Defense Medical Center, Taipei 11490, Taiwan
2
Graduate Institute of Biochemical and Biomedical Engineering, Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei 10608, Taiwan
3
Graduate Institute of Chemical Engineering, Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei 10608, Taiwan
biotechnological applications [5]. Dioxins and dioxin-like compounds, which are highly toxic environmental pollutants, bind the aryl hydrocarbon receptor, a protein enhancing certain transcriptions, affecting a number of other regulatory proteins to cause toxicity [2]. Instrument-based analytical methods including chromatography, such as high-performance liquid chromatography and gas chromatography, coupled mass spectrometry
Data Loading...
