Bioconjugation of aptamer to fluorescent trimethyl chitosan nanoparticles for bacterial detection
Introduction
Studies show that bacterial invasion of host barriers from the placental barrier to the blood-brain barrier (BBB) can lead to invasive infectious diseases [1]. While the pathway of the bacterial invasion is not understood yet. Therefore, it is highly needed to quickly detect bacteria in host barriers. Nucleic acid aptamers have shown the superior capability to specifically bind to analytes, including small molecules, proteins, whole-cell, bacteria [2], [3], [4], [5]. Since the development of the systematic evolution of ligands by exponential enrichment (SELEX) for produce aptamers for targets in 2012, more strategies have been explored to develop nucleic acid aptamers with a long half-life in vitro and in vivo [6]. In addition, chitosan is a natural polysaccharide and has been extensively used in drug delivery because of its good biocompatibility. Wheareas chitosan normally needs to be dissolved in an acidic solution due to its low solubility, which results in the limitation in loading medicines. N, N, N-trimethyl chitosan (TMC), a positively charged chitosan, has shown the promising capability for crossing tissue barriers, from mucosa to blood-brain barrier because it can interact with the tight junction proteins and reduce the electrical resistance of trans-epithelium [7], [8], [9]. Therefore, many efforts have been attempted to develop TMC nanoparticles (NPs) which has been used as a carrier to cross tissue barriers [10], [11]. Very few studies have been reported on integrating TMC NPs with nucleic acid aptamer for quick bacterial detection. Herein, fluorophore-labeled TMC NPs were synthesized by the microemulsion process in this paper. The single-strand DNA aptamer which has a strong affinity with E. coli (DH5α) was covalently bound on the surface of TMC NPs. The fluorescence intensity of TMC NPs as a function of the concentration of bacteria has been studied to quickly detect bacteria.
Section snippets
Materials
Ethyl oleate, 1,2-propanediol (PDO), chitosan, fluorescein isothiocyanate (FITC), glutaraldehyde (25%) and other chemicals were purchased from Sigma-Aldrich Chemical Co. Kolliphor® RH40 is a product from Basf Co.
Fluorophore loaded TMC NPs modified with aptamer
TMC product with 28% degree of quaternization was synthesized through the quaternization of chitosan (Fig. S1 in Supplementary Materials) [12]. To produce fluorescent TMC NPs, 5 mg fluorescein isothiocyanate (FITC) was dissolved in the oil phase with a mixture of 0.5 g ethyl oleate and
Results and discussion
Spherical TMC NPs can be observed in TEM micrographs as shown in Fig. 1a. The average particle size of TMC NPs is estimated at 323 ± 10 nm, which statistically measured by Image J®. Zeta potential (ζ) of the TMC NPs is measured at 37.1 ± 2.2 mV (Fig. S2 in Supplementary Materials). The large positive ζ-potential indicates those stable TMC NPs in terms of chemical properties have a net positive charge on the surface.
Aptamer (8.28A) 5′-NH2-(CH2)6-TCC TCG CGT TTG GAT TCA TGT TGG TTT GTC GGT GTA
Conclusion
28% degree of quaternization of chitosan was produced by using methyl iodide. The positively charged chitosan nanoparticles with an average diameter of 323 ± 10 nm were then successfully produced by a microemulsion process. The non-water-soluble organic dye is loaded in the nanoparticles with emission at 520 nm. Our result indicates TMC with the amine group can covalently bind to the nucleic acid aptamer, i.e. Aptamer (8.28A) by using the crosslinker, glutaraldehyde. A linear fitting curve is
Author contribution statement
J.Z., X.Z. conceived and designed the experiments. X.Z., L.C. performed major parts of experiments and analyzed the results. S.Y. performed all TEM measurements. The manuscript was written through the contributions of all authors.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
The authors are thankful for the financial support from the Natural Sciences and Engineering Research Council of Canada.
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