Bioprobes Based on Aptamer and Silica Fluorescent Nanoparticles for Bacteria Salmonella typhimurium Detection
© Wang and Kang. 2016
Received: 16 November 2015
Accepted: 7 March 2016
Published: 16 March 2016
In this study, we have developed an efficient method based on single-stranded DNA (ssDNA) aptamers along with silica fluorescence nanoparticles for bacteria Salmonella typhimurium detection. Carboxyl-modified Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate (RuBPY)-doped silica nanoparticles (COOH-FSiNPs) were prepared using reverse microemulsion method, and the streptavidin was conjugated to the surface of the prepared COOH-FSiNPs. The bacteria S. typhimurium was incubated with a specific ssDNA biotin-labeled aptamer, and then the aptamer-bacteria conjugates were treated with the synthetic streptavidin-conjugated silica fluorescence nanoprobes (SA-FSiNPs). The results under fluorescence microscopy show that SA-FSiNPs can be applied effectively for the labeling of bacteria S. typhimurium with great photostable property. To further verify the specificity of SA-FSiNPs out of multiple bacterial conditions, variant concentrations of bacteria mixtures composed of bacteria S. typhimurium, Escherichia coli, and Bacillus subtilis were treated with SA-FSiNPs.
In addition, the feasibility of SA-FSiNPs for bacteria S. typhimurium detection in chicken samples was assessed. All the results display that the established aptamer-based nanoprobes exhibit the superiority for bacteria S. typhimurium detection, which is referentially significant for wider application prospects in pathogen detection.
Bacteria Salmonella typhimurium (bacteria S. typhimurium) is a kind of food-borne zoonotic pathogen, which may cause food poisoning, acute gastroenteritis, and exogenous febrile disease and even lead to death [1, 2]. Especially, food poisoning caused by bacteria S. typhimurium is at the top of the bacterial food poisoning event list in the world . Generally, bacteria S. typhimurium is mainly detected using morphological observation, immunological method, and molecular techniques nowadays [4–6]. Briefly, the morphological observation method only provides the shape, size, and dyeing property (gram positive or gram negative) of bacteria, which cannot provide the specific types of information of the bacteria the researchers look for. Although the immunological method, like enzyme-linked immunosorbent assay (ELISA), is a widely used method for bacteria detection, the procedure is complex and time-consuming. The PCR-based methods are popular at present, but they are easily contaminated and time-consuming sometimes. Therefore, to develop a fast and effective method for bacteria S. typhimurium detection to remedy the extant weaknesses is essential in a pathogen-monitoring program as well as clinic diagnosis.
In recent years, the aptamer has attracted tremendous interest because of its excellent features for pathogen detection. Aptamers are kinds of synthetic peptide or oligonucleotide molecules which can bind to the target molecule firmly. With advantage of high affinity and specificity, the aptamers are prominent for detecting all kinds of targets, such as tumor cells, bacteria, virus, proteins, some small molecules (ATP), and even metal ions [7–11]. Compared with antibodies, aptamers would be promising recognition elements for bioanalysis because of their high affinity and specificity with various kinds of targets, easy preservation, facile modification, and good stability.
For the application in bacteria S. typhimurium detection, several aptamer probes had been selected and characterized using whole-bacterium SELEX [12–14]. According to the structural analysis of a selected aptamer and measurement of affinity constant between the aptamer and bacteria S. typhimurium, the bacteria S. typhimurium aptamer is a 40-base single-stranded DNA with Kd value (6.33 ± 0.58 nM), which indicates the high affinity and specificity of aptamers . In addition, the single-stranded oligodeoxynucleotide anti-S. typhimurium aptamer has a lower molecular weight compared to antibodies.
The fluorescent dyes are materials commonly used as an indicator in bioanalysis, while the deficiency of photobleaching severely hinders the range of their applications. With the development of nanotechnology in recent years, fluorescent nanomaterials play an increasingly important role in bioanalysis . Many researches had revealed that the nanometer material shell surrounding the dye molecule could prevent the dye from photobleaching. In addition, a larger amount of dye molecules entrapped inside the silica matrix would play the role of a signal amplifier. Then, the fluorescent core-shell nanoparticles, like the dye-doped silica nanoparticles, have attracted great attention due to their other good characters with respect to photostability, surface-to-volume ratio, modification, cost, biocompatibility, and hydrophilicity [16–19]. For instance, targeted fluorescence nanoprobes are prepared by conjugating the desirable biomolecules (antibody or protein) to the surface of the dye-doped silica nanoparticles, which have been widely used for tumor cell recognition and separation, bacteria labeling, and DNA ultrasensitive assay [20–22].
Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate (RuBPY) is a kind of hydrophilic positively charged fluorescent dye, which shows the fluorescence emission wavelength (610 nm) with the excitation wavelength at 450 nm. As mentioned above, compared with the free RuBPY dye, the RuBPY-doped silica nanoparticles take advantage of resistance to photobleaching due to dye molecules with a positive charge entrapped stably inside the negatively charged silica matrix, which prevents the potential quenching substance approaching the dye molecules [23, 24].
In view of the extraordinary properties of aptamer and fluorescent nanomaterials for target labeling and signaling [25–28], we prepare novel bioprobes based on single-stranded DNA (ssDNA) aptamers and RuBPY-doped silica nanoparticles for bacteria S. typhimurium detection, of which the availability was accessed in a variety of conditions.
Triton X-100, tetraethyl orthosilicate (TEOS), (3-aminopropyl)triethoxysilane (APTES), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS), RuBpy, and streptavidin were purchased from Sigma Chemical Co. (St. Louis, MO). Ammonium hydroxide (25–28 wt%), n-hexanol, cyclohexane, acetone, alcohol, N,N-dimethylformamide (DMF), and succinic anhydride were purchased from China Pharmaceutical Group Shanghai Chemical Reagent Co., Ltd. Bacteria S. typhimurium, Escherichia coli (E. coli) DH5ɑ, and Bacillus subtilis (B. subtilis) were obtained from China Center for Type Culture Collection. The rhodamine B isothiocyanate (RBITC)-labeled bacteria S. typhimurium aptamer was purchased from Beijing Biosynthesis Biotechnology Co., Ltd. The biotin-labeled bacteria S. typhimurium aptamer: 5′-biotin-(CH2)6-AGTAATGCCCGGTAGTTATTCAAAGATGAGTAGGAAAAGA-3′ and the biotin-labeled random sequence: 5′-biotin-(CH2)6-TGTCATGACCCGTAGGTAGTCTTAGAAGACTAGGCACGTT-3′ were synthesized at Shanghai Sangon Biological Engineering Technology & Services Co. (China).
The size and uniformity of synthesized silica NPs were measured by means of transmission electron microscopy (JEOL, JEM100CXII, Japan). Photoluminescence was measured using a fluorescence spectrophotometer (F96PRO, China). Fluorescence image results were observed under an inverted fluorescence microscope (Nikon ECLIPSE TE2000-U, Japan).
Preparation and Characterization of Streptavidin-Conjugated RuBPY-Doped Silica Fluorescence Nanoprobes
Carboxyl-modified RuBPY-doped silica nanoparticles (COOH-FSiNPs) were synthesized by the method described by Arriagada et al., and the detailed procedure referenced to Cai et al. [29, 30]. In short, the RuBPY-doped silica NPs were prepared through the polymerization reaction of TEOS and NH4OH with RuBpy in the water-in-oil microemulsion, which was made of Triton X-100, cyclohexane, and water. Then, the RuBPY-doped silica was amine modified using TEOS and APTES. Afterwards, the generated floccule was deposited using EDC and Sulfo-NHS and washed with acetone after centrifugation. By washing and resuspending with DMF solution, the products were reacted with succinic anhydride under nitrogen gas for 24 h with continuous stirring. Then, the prepared COOH-FSiNPs were washed with water and resuspended in PBS for the next step.
Two milligrams of COOH-FSiNPs, 1 mg of EDC, and 2.5 mg of Sulfo-NHS were added to 1 mL of 0.1 M PBS buffer (pH 7.4). The mixture was then incubated for 15 min at room temperature with gentle shaking. After the reaction completed, 50 μL of streptavidin diluted in PBS at a concentration of 1 mg/mL was immediately added to the solution, following a 3-h incubation with gentle shaking at room temperature. The particles were washed with 0.1 M PBS (pH 7.4) and then resuspended in 1 mL of 0.05 % BSA for 1 h to block the free carboxylates. After the reaction completed, the prepared streptavidin-conjugated silica fluorescence nanoprobes (SA-FSiNPs) were washed with 0.1 M PBS buffer (pH 7.4) three times and then resuspended in 0.1 M PBS buffer (pH 7.4) for further use.
Application of the Nanoprobes for Bacteria S. typhimurium Detection
One hundred microliters of 10 μM biotin-labeled anti-S. typhimurium aptamer and 1 mL of bacteria S. typhimurium diluted in PBS at a concentration of 80 cfu/mL were incubated for 60 min at 37 °C with gentle shaking. After the reaction completed, the suspension of bacteria S. typhimurium aptamer complex was centrifuged and washed with PBS three times and then resuspended in 1 mL of 0.1 M PBS (pH 7.4). One hundred microliters of SA-FSiNPs was immediately added to the suspension. After incubation for 60 min at 37 °C with gentle shaking, the mixtures were centrifuged (6000 rpm × 5 min) and washed with 0.1 M PBS (pH 7.4) three times to remove the free SA-FSiNPs. The probe-bacteria conjugates were finally resuspended in PBS and then smeared on a slide glass for fluorescence microscope observation. The corresponding control group was treated through the same procedure with exception of the anti-bacteria S. typhimurium aptamer which was replaced by a biotin-labeled random sequence aptamer.
Streptavidin-Conjugated Silica Fluorescence Nanoprobe for Bacteria S. typhimurium Detection Under Multiple Bacterial Conditions
Group A: 80 cfu/mL bacteria S. typhimurium + 40 cfu/mL E. coli DH5ɑ + 20 cfu/mL B. subtilis
Group B: 40 cfu/mL bacteria S. typhimurium + 20 cfu/mL E. coli DH5ɑ + 80 cfu/mL B. subtilis
Group C: 20 cfu/mL bacteria S. typhimurium + 80 cfu/mL E. coli DH5ɑ + 40 cfu/mL B. subtilis
The three groups were incubated with 300 μL of 10 μM biotin-labeled anti-S. typhimurium aptamer for 60 min at 37 °C with gentle shaking. After the reaction completed, three kinds of bacteria mixture were centrifuged and washed with PBS three times and then resuspended in 1 mL of 0.1 M PBS (pH 7.4). Two hundred microliters of SA-FSiNPs was immediately added to the bacteria mixture. Subsequent steps were as described in the “Application of the Nanoprobes for Bacteria S. typhimurium Detection” section.
Fluorescence Nanoprobe for the Bacteria Detection in Chicken Samples
Five grams of minced chicken samples purchased from a supermarket and 100 μL of 1 × 103 cfu/mL bacteria S. typhimurium were mixed in the triangular flask containing 50 mL of bacteria culture media (Mueller Hinton Broth). The mixture was then incubated for 12 h at 37 °C with gentle shaking. After the reaction completed, the culture media were centrifuged (1000 rpm × 5 min) and washed with PBS three times. The chicken samples were resuspended in 10 mL of 0.1 M PBS (pH 7.4). One hundred microliters of 10 μM biotin-labeled bacteria S. typhimurium aptamer was then added to the chicken samples dispersed in PBS. After incubation for 60 min at 37 °C with gentle shaking, the chicken sample suspension was centrifuged (5000 rpm × 5 min) with PBS three times. The chicken samples were then resuspended in 5 mL of 0.1 M PBS (pH 7.4). Two hundred microliters of SA-FSiNPs was subsequently added and then incubated for 2 h at 37 °C with gentle shaking. After centrifugation (1000 rpm × 5 min) and washing, the supernatant was transferred into a centrifuge tube and centrifuged (5000 rpm × 5 min). The probe-bacteria conjugates were finally resuspended in PBS and then smeared on a slide glass for fluorescence microscope observation. The procedures corresponding to the experimental groups were performed in the control groups except that the ground chicken samples were incubated with bacteria culture media in absence of bacteria S. typhimurium.
Quantitative Analysis of Bacterial Cell Staining
Quantification of bacterial cell staining was performed using ImageJ software according to its standard manual.
Results and Discussion
Characterization of Carboxyl-Modified RuBPY-Doped Silica Nanoparticles
Labeling and Imaging for Aptamer-Based Bacteria S. typhimurium Detection
The Specificity Verification of SA-FSiNPs
Photostability of Streptavidin-Conjugated Silica Fluorescence Nanoprobes
Application of the Bacteria S. typhimurium Detection out of Chicken Samples
A simple but effective method based on biotin-labeled aptamer and streptavidin-conjugated silica fluorescence nanoprobes for bacteria S. typhimurium detection had been established. The biotin-labeled anti-bacteria S. typhimurium aptamer shows high selectivity and affinity to membrane proteins of this food-borne pathogen. Because of the specific binding between streptavidin and biotin, the SA-FSiNPs can be effectively applied for the labeling of bacteria S. typhimurium in various complex backgrounds, which had been further confirmed in the multiple bacteria mixture and practical chicken samples. In addition, SA-FSiNPs display good photostability than the dye-labeled probes. Moreover, silica nanomaterials take advantage of good biocompatibility, which can serve as a necessary and useful supplement for the current detection methods.
This work was financially supported by the Fundamental Research Funds for the Central Universities (No. JUSRP11568), a grant from Public Health Research Center at Jiangnan University (No. JUPH201505), and a grant from Jiangsu Science and Technology Department (No. BM2015024-3).
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