Abstract
In this paper, a fast and simple strategy for sensitive detection of streptavidin (SA) was proposed based on terminal protection of small molecule-linked DNA and cationic conjugated polymer-mediated fluorescence resonance energy transfer (FRET). In principle, we designed a biotin-labelled DNA probe (P1) as the recognitive probe of SA, along with a complementary DNA probe (P2) to form double-stranded DNA (dsDNA) with P1. SYBR Green I (SG I) as a fluorescent dye was further used to specifically bind to dsDNA to emit stronger fluorescence. The cationic poly[(9,9-bis(6′-N,N,N-triethy-lammonium)hexyl) fluorenylene phenylene dibromide] (PFP) acted as the donor to participate in the FRET and transfer energy to the recipient SG I. In the absence of SA, P1 could not hybridize with P2 to form dsDNA and was digested by exonuclease I (Exo I); thus, only a weak FRET signal would be observed. In the presence of SA, biotin could specifically bind to SA, which protected P1 from Exo I cleavage. Then, P1 and P2 were hybridized into dsDNA. Therefore, the addition of SG I and PFP led to obvious FRET signal due to strong electrostatic interactions. Then, SA can be quantitatively detected by monitoring FRET changes. As the whole reagent reaction was carried out in 1.5 mL EP and detected in the colorimetric dish, the operation process of the detection system was relatively simple. The response time for each step was also relatively short. In this detection system, the linear equation was obtained for SA from 0.1 to 20 nM with a low detection limit of 0.068 nM (S/N = 3). In addition, this strategy has also achieved satisfactory results in the application of biological samples, which reveals the application prospect of this method in the future.
Highlights
Streptavidin (SA) is a 58.2 kDa protein secreted by the bacterium Streptomyces avidinii and composed of four identical peptide chains contains tryptophan, which can bind to biotin with high specificity and strong affinity [1,2]
Streptavidin (SA), Carcinoembryonic antigen (CEA), alpha fetoprotein (AFP) and Immunoglobulin G (IgG) were all obtained from Sigma-Aldrich
The SA–biotin system composed of SA and biotin can bind to antigens, antibodies, enzymes, oligonucleotide molecules and fluorescent substances, and has a wide range of applications in biology, especially in the detection of immune detection, antigen, antibody
Summary
Streptavidin (SA) is a 58.2 kDa protein secreted by the bacterium Streptomyces avidinii and composed of four identical peptide chains contains tryptophan, which can bind to biotin with high specificity and strong affinity [1,2]. Because of the specific interactions of SA and biotin, the development of a highly sensitive SA detection strategy is very beneficial for applications in disease prediction, chemical genetics, and drug development [6]. An increasing number of methods have been developed for SA detection and quantitative analysis, including protein-fragment complementation assay [7], affinity chromatography [8], kinetic capillary electrophoresis [9] and surface plasmon resonance mboxciteB7-polymers-1116075,B10-polymers-1116075. Most of these traditional methods have disadvantages such as small samples, expensive instruments, time-consuming detection and cumbersome operation processes, which greatly limit their application [11]
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