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Aptamer-based hydrogel barcodes for the capture and detection of multiple types of pathogenic bacteria
Biosensors and Bioelectronics 100 (2018) 404–410
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Aptamer-based hydrogel barcodes for the capture and detection of multiple types of pathogenic bacteria ⁎
Yueshuang Xua, Huan Wangb, Chengxin Luana, Yuxiao Liub, Baoan Chena, , Yuanjin Zhaoa,b, a b
MARK
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Department of Hematology and Oncology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Inverse opal Barcode Bacteria Aptamer Magnetic properties
Rapid and sensitive diagnosing hematological infections based on the separation and detection of pathogenic bacteria in the patient's blood is a significant challenge. To address this, we herein present a new barcodes technology that can simultaneously capture and detect multiple types of pathogenic bacteria from a complex sample. The barcodes are poly (ethylene glycol) (PEG) hydrogel inverse opal particles with characteristic reflection peak codes that remain stable during bacteria capture on their surfaces. As the spherical surface of the particles has ordered porous nanostructure, the barcodes can provide not only more surface area for probe immobilization and reaction, but also a nanopatterned platform for highly efficient bioreactions. In addition, the PEG hydrogel scaffold could decrease the non-specificity adsorption by its anti-adhesive effect, and the decorated aptamer probes in the scaffolds could increase the sensitivity, reliability, and specificity of the bacteria capture and detection. Moreover, the tagged magnetic nanoparticles in the PEG scaffold could impart the barcodes with controllable movement under magnetic fields, which can be used to significantly increase the reaction speed and simplify the processing of the bioassays. Based on the describe barcodes, it was demonstrated that the bacteria could be captured and identified even at low bacterial concentrations (100 CFU mL−1) within 2.5 h, which is effectively shortened in comparison with the “gold standard” in clinic. These features make the barcodes ideal for capturing and detecting multiple bacteria from clinical samples for hematological infection diagnostics.
1. Introduction Bacteremia caused by bacterial bloodstream infections, such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and so on, can lead to vascular leakage, tissue damage, and multiorgan failure; these are associated with one-third of global mortality (Angus and van der Poll, 2013; Kailasa and Wu, 2012, 2013; Rocheteau et al., 2015). Identifying bacteria rapidly in the early stage of infection is significant to decrease high mortality (Yealy et al., 2014). However, current “gold standard” in clinical diagnosis of bacteremia usually needs 3–5 days of incubation and at least 12 h of growing on solid media to identify the bacteria (Sarkar et al., 2006). Various approaches have been devised to improve the sensitivity for bacterial identification, such as real-time polymerase chain reactions (Ottesen et al., 2006), fluorescent in situ hybridization, surface enhanced Raman scattering, and fluorescent probes (Kang et al., 2014). In spite of the improvement, these methods still require long-term blood culture and expensive equipment, which limit their widespread use in clinical applications. As an alternative,
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some simple cell capture platforms have been constructed to shorten the time needed for pathogen detection as well as identification. However, most of these methods were only carried out with single target test, and could not effectively distinguish between different bacteria simultaneously in a simple detection. Therefore, the development of a new platform that can capture and distinguish multiple types of bacteria simultaneously in a short time is highly desired. Barcodes(also called as encoded microcarriers), which encode information about their specific compositions and enable simple identification, have attracted increasing interest for multiple bioassays (Liu et al., 2014; Meng et al., 2015; Shi et al., 2013; Xu et al., 2017; Yang et al., 2008; Zhang et al., 2016; Zheng et al., 2014). Many kinds of encoding strategies have been proposed for the barcodes, including fluorescent molecules, quantum dots, photonic crystals, or graphical or shape-encoded microplates, and so on (Ge and Yin, 2008, 2011; Kanai et al., 2010; Lee et al., 2015; Mao et al., 2010; Shang et al., 2013; Sim et al., 2015; Song et al., 2015; Xu and Chen, 2015; Yu et al., 2009; Y.Q. Zhang et al., 2013, Y.S. Zhang et al., 2013; Zhao et al., 2014, 2015).
Corresponding author. Corresponding author at: Department of Hematology and Oncology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China. E-mail addresses: [email protected] (B. Chen), [email protected] (Y. Zhao).
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http://dx.doi.org/10.1016/j.bios.2017.09.032 Received 30 July 2017; Received in revised form 12 September 2017; Accepted 18 September 2017 Available online 20 September 2017 0956-5663/ © 2017 Elsevier B.V. All rights reserved.
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Scheme 1. Schematic diagram of the inverse opal structured magnetic hydrogel barcodes with aptamer probes for the bacteria capture.
hydroxy-2-methylpropiophenone (HMPP) photoinitiator were purchased from Sigma-Aldrich, Shanghai, China. Acrylic Acid (AA) was obtained from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Aladdin Reagent Co., Ltd., Shanghai, China. 2-Morpholinoethanesulfonic Acid (MES) was obtained from AMRESCO LLC, Solon, USA. Phosphate buffer saline (PBS, 0.05 M, pH 7.4) were self-prepared. All buffers were self-prepared using water purified in a Milli-Qsystem (Millipore, Bedford, USA). 30 healthy human blood samples were collected from the affiliated ZhongDa Hospital of Southeast University, Nanjing, China. All the collection and processing of human blood samples were carried out in accordance with the guidelines issued by the Ethical Committee of the Chinese Academy of Sciences.
Based on these barcodes, many distinct multiple assays have been carried out for high throughput biomolecule detection, gene function analysis, and clinical diagnosis (Fu et al., 2016). However, the encoded information of most barcodes would be confused or incomprehensible when their surfaces were covered by bacteria, and this could cause false decoding of the barcodes. In addition, the debatable specificity and reliability of their general surface morphology and biochemical modification, as well as the uncontrolled motion, have also limited the application of barcodes for the bacteria detection. Thus, the development of new barcodes-based bacteria capture and detection platform with distinct advantages is still required. Scheme 1 In this paper, we present a new aptamer-functionalized barcodes technology that can simultaneously capture and detect different types of pathogenic bacteria. The barcodes are poly (ethylene glycol) (PEG) hydrogel inverse opal particles with characteristic reflection peak codes that remain stable during bacteria capture on their surfaces (Choi et al., 2009; Hong et al., 2012; Shang et al., 2015; Stein et al., 2013; Y.Q. Zhang et al., 2013, Y.S. Zhang et al., 2013). The decorated aptamer probes in the PEG scaffolds could specifically capture the bacteria, while the PEG hydrogel scaffold could decrease the non-specificity adsorption of other targets. Due to the ordered porous nanostructure on the spherical surface, the barcodes can provide not only more surface area for probe immobilization and reaction, but also a nanopatterned platform for highly efficient bioreactions. In addition, the tagged magnetic nanoparticles in the PEG scaffold could impart the barcodes with controllable movement under magnetic fields, which can be used to significantly increase the reaction speed and simplify the processing of the bioassays. Thus, compared with other reported methods, our barcodes have such advangtages, include low non-specificity adsorption, good efficiency, fast capture speed and multiplex capture capacity. It will be demonstrated that the bacteria with low concentrations could be captured and identified very fast by the barcodes, which fully satisfied the clinical criteria during the hematological infection diagnostics.
2.2. Instruments The microfluidic device used for generating silica colloidal crystal beads was home-made. All reactions were finished in flat-bottom tubes and a constant temperature shaker (Thermomixer comfort 5355, Eppendorf, Germany). The microstructures of silica colloidal crystal beads and hydrogel photonic barcodes were characterized according to a scanning electron microscope (SEM, S-300N, Hitachi, Japan). Photographs of the two kinds of beads were taken by an optical microscope (BX51, Olympus, Japan) equipped with a CCD camera (MP5.0, Media Cybernetics Evolution). The reflectance spectra of the barcodes were recorded by the same microscope equipped with a fiber optic spectrometer (HR2000, Ocean Optics, USA). The fluorescence intensity was detected by a fluorescence microscope (BX53, Olympus, Japan). 2.3. Fabrication of inverse opal hydrogel magnetic barcodes Silica colloidal crystal beads (SCCBs) used as the template to fabricate this kind of inverse opal barcodes were fabricated using the microfluidic devices. The inverse opal barcodes were replicated from the voids of the template silica colloidal crystal beads. And the pre-gel solution used for the fabrication of inverse opal barcodes was composed of Poly (ethyleneglycol) diacrylate (PEG-DA) and Acrylic Acid (AA). Firstly, the dried silica colloidal crystal beads with different colors were immersed in pre-gel solution (20% PEG-DA, 10%AA and 1% HMPP) for 1 h. The liquid mixed solution could fill the gaps of silica colloidal crystal beads fully. Next, the mixture of beads and pre-gel solution was exposed to UV light for polymerizing the pre-gel solution in and out of the SCCBs. After polymerization, the hybrid beads of different colors could be extracted by stripping the pre-gel on the surface of the beads, and then remove the silica template with 4% hydrofluoric acid to obtain hydrogel inverse opal barcodes. Finally, the magnetic inverse opal barcodes could be obtained by saturating the barcodes with the magnetic nanoparticles.
2. Experimental section 2.1. Materials Six kinds of SiO2 nanoparticles with the size of 211, 260 and 307 nm were purchased from NanJing DongJian Biological Technology Co., Ltd. CY3 labeled rabbit polyclonal anti-human AFP antibody were purchased from Micro Biological Technology Company, Shanghai, China. Two kinds of aptamers (AptS.aureus and AptE.coli) were purchased from Ruibo Biological Technology Co., Ltd., Guangzhou, China. Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) were purchased from the inquiry network for microbial strains of China. FITC Concanavalin A was purchased from Sigma-Aldrich, Shanghai, China. Poly (ethyleneglycol) diacrylate (PEG-DA) with molecular weights of 700 and 2405
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Fig. 1. (a) The optical images of the monodisperse hydrogel barcodes; (b) reflection spectra and optical images of six kinds of hydrogel barcodes; (c) hydrogel barcodes; (d) hydrogel barcodes under a magnetic field. Scale bar is 200 µm.
reaction process, we use magnet to shuttle the barcodes in the samples constantly. Then wash away the residual infected blood around the barcodes twice with PBS buffer. The efficiency of detection is evaluated through fluorescence intensity of the barcodes. The method for detecting S.aureus is the same as that for E.coli. To validate the feasibility of applying magnetic hydrogel inverse opal barcodes in capturing multiple types of bacteria in blood, three kinds of magnetic inverse opal barcodes with characteristic reflection peaks at 600, 530, and 440 nm (referred to as red, green, and blue, respectively) were used in the experiment for multiple bacteria capture. For this purpose, the red inverse opal barcodes were coated with AptS.aureus, and the green inverse opal barcodes were with AptE.coli, while the blue one served as a control with no aptamer adsorption. These barcodes were then mixed and incubated in blood samples spiked with S.aureus or E.coli which were dyed with FITC Concanavalin A for 2.5 h at 37 °C with constant shuttle. Then wash away the residual infected blood around the barcodes twice with PBS buffer and observe the specificity of this experiment through fluorescence intensity of the barcodes. Subsequently, to image the samples by using a scanning electron microscope, the captured bacteria were fixed using 4% (v/v) paraformaldehyde overnight at 4 °C followed with dehydration with gradient ethanol gradually. The common concentration gradient of ethanol is from 20%, 40%, 60%, 80–100%, and the samples were dehydrated for over 20 min in each concentration.
2.4. Preparation of the aptamer-conjugated inverse opal hydrogel magnetic barcodes Two kinds of aptamer probes (AptS.aureus and AptE.coli) were dissolved in sterile water at the concentration of 10 mM. Firstly, treat the magnetic hydrogel inverse opal barcodes prepared before with 2Morpholinoethanesulfonic Acid (MES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for 30 min at 37 ℃ in the constant shaker. Then red inverse opal barcodes were coated with amino-modified AptS.aureus, and green barcodes were coated with amino-modified AptE.coli after being washed with PBS for 6 h at 25 ℃. Finally, the different functionalized inverse opal barcodes with own probes were prepared successfully after washing with buffer solution. 2.5. Bacterial sample culture The Luria−Bertani (LB) broth was prepared by dissolving 10 mg/ mL tryptone, 5 mg/mL yeast extract and 10 mg/mL Nacl in purity water followed with sterilization. The strains of S. aureus and E. coli were cultured with LB broth at 37 °C overnight, and the colony forming units (CFUs) were determined by measuring the optical density (OD) at 600 nm (OD600 = 1.0 is approximately 1.0 × 109 CFU mL−1). In detail, we first diluted the culture to obtain an appropriate OD value of about 1.0. After that, the bacterial solution with an OD600 value of about 1.0 was serially diluted and inoculated onto the solid medium to quantify the CFU mL−1.
3. Results and discussion 3.1. Design of the hydrogel barcodes
2.6. Selectivity Test of S. aureus and E.coli As a typical experiment, the inverse opal structured hydrogel barcodes were fabricated by replicating silica colloidal crystal bead templates. These bead templates were prepared by the ordered self-assembly of silica nanoparticles in microfluidic droplets during the evaporation of water. The ordered structure of the nanoparticles gives the colloidal crystal beads brilliant structural colors and reflection spectra (Fig. S1). To generate the hydrogel barcodes, the silica colloidal crystal bead templates were first immersed in a pre-gel solution. After the pre-gel solution had filled the voids between the nanoparticles of
First of all, we dyed the bacteria with FITC Concanavalin A under protection from light for half an hour. Then, the bacteria were washed three times with PBS buffer by centrifugation to remove free FITC Concanavalin A. In a typical experiment, human blood was spiked with E.coli to simulate bacteremia at a final concentration of 104 CFU mL−1. Subsequently, almost ten aptamer-conjugated magnetic inverse opal barcodes were added to 100 μL infected blood, and the mixture was incubated for 2.5 h followed by magnetic separation. In this whole 406
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Fig. 2. SEM images: (a, b) surface structure of the colloidal crystal beads, which indicated a commendable hexagonal close packing structure; (b) inverse opal barcodes with an interconnected porous surface; (c) the captured E.coli on the surface of the AptE.coli-decorated inverse opal hydrogel barcodes; (d) the captured S.aureus on the surface of the AptS.aureus-decorated inverse opal hydrogel barcodes. Scale bars are 500 nm in (a-b), 2 µm in (c) and 500 nm in (d).
morphology on their surfaces (Fig. 2d). It was worth to mention that when the bacteria were captured on the barcodes, they did not affect the ordered structure of the whole sphere. Thus, the encoded elements of the inverse opal barcodes remained constant during the bacteria capture. Due to the ordered porous nanostructure, the hydrogel barcodes were imparted with a photonic band gap (PBG) property and show the corresponding structural color or characteristic reflection peak. Under normal incidence, the peak positions λ of the barcodes can be estimated by Bragg's equation [Eq. (1)]
the templates by the capillary force, the pre-gel solution in and out of the bead templates was polymerized by using ultraviolet light. Finally, the inverse opal structured hydrogel barcodes were obtained by removing the pre-gel on the surface of the templates and etching the silica nanoparticles in the templates with hydrofluoric acid (Fig. 1). To impart the resultant barcodes additional feature, an ultrafine magnetic nanoparticles solution was employed for immersing the inverse opal structured hydrogel barcodes. This treatment imparted the barcodes with controllable movement under magnetic fields (Fig. 1c, S1c-d), which was potential to increase the reaction speed and simplify the processing of the bioassays. The microstructures of the inverse opal structured magnetic hydrogel barcodes and their templates were observed with a scanning electron microscope (SEM). It can be seen from Fig. 2 that the silica nanoparticles on the surface of the template beads mainly formed a hexagonal alignment (Fig. 2a). Thus, the inverse opal barcodes replicated from the templates should have a similar highly ordered three dimensional (3D) inverse opal structure and hexagonal symmetrical porous surface. As expected, it can be observed that the barcodes had an interconnected and hexagonal symmetrical porous surface (Fig. 2b), which would provide a nanopatterned platform for highly efficient entrapping bacteria. To capture bacteria, different types of probes could be employed. Among these probes, aptamers are single stranded DNA or RNA oligonucleotides that can bind to variety of targets with high selectivity and strong affinity. Compared to other biomolecules, aptamers have some advantages, including small size, easy modification, and synthesis. Recently, several bacterial aptamers with high selectivity have been developed, so they are ideal options to detect specific bacteria from blood samples. As a proof of concept in this work, we used AptS.aureus or AptE.coli (Fig. S2, and the aptamer sequences were in Table S1) to capture S.aureus or E.coli, respectively. To confirm their ability, the AptE.coli-decorated barcodes were incubated in E.coli suspension and observed under SEM after the reaction. Fig. 2c showed that large numbers of E.coli were captured on the surface of the inverse opal barcodes, and the normal morphology of E.coli was maintained. Similarly, the AptS.aureus-decorated barcodes captured several S.aureus with normal
λ = 1.633dnaverage
(1)
where d is the center-to-center distance between two neighboring nanopores, and naverage is the average refractive index of the barcodes. Therefore, by using different sizes of silica nanoparticles assembled templates, a series of hydrogel barcodes with different sizes of nanopores and reflection peaks could be obtained for the coding (Fig. 1b). As there was no dye or other materials related with the code, concerns about chemical instability and the fluorescence background during the bacteria capture and detection were not necessary. 3.2. Optimization of the material To reduce the nonspecific adsorption of the proteins and other cells to the barcodes during the bacteria capture and detection, the PEG hydrogel, which has the advantages of anti-adhesive effect and mechanical strength, was employed as the scaffold material of the barcodes. In addition, in order to immobilize the amino-modified aptamers on the surface of the barcodes, the acrylic acid with a large number of carboxyl was also used as the hydrogel component. Thus, the pre-gel solution consisted of the poly(ethylene glycol) diacrylate (PEG-DA), acrylic acid and 2-hydroxy-2-methylpropiophenone photoinitiator. In general, lower concentration of PEG-DA could offer larger grid to enhance the actives of the immobilized aptamer probes. To confirm this, five kinds of inverse opal barcodes with different concentration of PEGDA were fabricated and immobilized with probes (Fig. S3). Then these barcodes were incubated in the fluorescence labeled targets. It was 407
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Fig. 3. Laser scanning confocal microscopy (LSCM) images of the captured E.coli bacteria by different hydrogel barcodes: (a) hydrogel barcodes without AptE.coli probes; (b) hydrogel barcodes with AptE.coli probes; (c) magnetic hydrogel barcodes with AptE.coli probes. From left to right: cross-section fluorescent images, merged cross-section fluorescent and white-light images, and 3D fluorescent images. Scale bar is 50 µm.
found that the fluorescence intensity of the barcodes with lowest PEGDA concentration (20%) was higher than those with 40%, 60% and 80% PEG-DA concentrations (Fig. S4), this indicated much targets were captured. Although a much higher capture performance would be achieved when the concentration of PEG-DA was lower than 20%, the barcodes showed poor mechanical properties. Thus 20% PEG-DA was used to fabricate all the hydrogel barcodes for the following experiments. It was worth to mention that the fluorescence intensity was pretty low for the control barcodes group that without aptamer probes immobilization, which confirmed the anti-adhesive effect of the PEG hydrogel material.
without AptE.coli probes, hydrogel barcodes with AptE.coli probes, and magnetic hydrogel barcodes with AptE.coli probes, respectively (Fig. 3). It can be seen that the latter two kinds of barcodes with aptamer probes can capture more bacteria, which means that the aptamer probes with strong affinity can surely increase the capture specificity. In addition, because the magnetic barcodes was shuttled in the samples constantly with the controllable movement under magnetic fields (Fig. 1c, Fig. S5), they could capture more bacteria on their surfaces than those barcodes without controllable movement, as shown in Fig. 3b and c. To further increase the capture efficiency of the bacteria on the magnetic hydrogel barcodes, we have also optimized the concentrations of the aptamer probes and the incubation times during the capture (Fig. 4a). In these processes, the barcodes were decorated with different concentrations of AptE.coli (50, 100, 200, 500, 1000and 2000 μM) for the probes immobilization, and then these barcodes were added into the same concentration of E.coli for the same capture time. It was found that with the increasing of the concentration of the aptamer, the fluorescence intensity of the captured bacteria on the barcodes gradually increased, and they saturated when the concentration exceeded
3.3. Optimization of the reaction conditions To visually observe the captured bacteria on the surface of the barcodes, E.coli were stained by using FITC Concanavalin A for 30 min at room temperature. After this staining, a laser scanning confocal microscopy (LSCM) was employed for the cross-section and three-dimensional images of the E.coli on the surface of the hydrogel barcodes 408
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we chose to use the capture time of 3.5 h to ensure nearly complete capture in this work for faster detection. This capture time indicated a much faster detection process than the previous method in clinic, and this advantage may realize the rapid diagnosis of bacteria in future.
3.4. Bacteria capture For the optimized experiments mentioned above, the bacteria were designed with high concentrations in the blood samples for the researches. However, the infectious dose of bacteria in the real blood sample was usually very low. Thus, to evaluate the capture sensitivity of the magnetic hydrogel barcodes at low bacterial concentrations, human blood samples were also spiked with different concentrations of E.coli (100, 500, 1000, 2500, 5000, and 104 CFU mL−1) for the barcodes detection. The results showed that the numbers and the relative fluorescence intensities of E.coli on the barcodes increased with the increasing of the concentration of the E.coli in the blood samples (Fig. 4b and Fig. S6). The fluorescence intensity of the background was 17, and the standard deviation was 10, so it is noteworthy that the bacteria could be detected even at a low concentration of 100 CFU mL−1; this indicated the feasibility of the magnetic hydrogel barcodes for bacteria detection in clinical blood samples. To demonstrate the specificity of different aptamers and the capability of the magnetic hydrogel barcodes for capturing multiple types of bacteria in blood, three kinds of magnetic barcodes with characteristic reflection peaks at 600, 530, and 440 nm (referred to as red, green, and blue, respectively) were used for the test, as shown in Fig. 5. In this experiment, the red and green barcodes were decorated with AptS.aureus and AptE.coli, respectively; while the blue one served as a control without aptamer probe immobilization. These barcodes were then mixed together and incubated in healthy human blood spiked with low concentration of S.aureus. It was found that S. aureus were mainly captured on the red barcodes due to the specific binding between the S.aureus and the AptS.aureus, while the green and blue barcodes showed no obvious capture (Fig. 5c). By using E.coli to instead S.aureus in the blood, it could got an inverse result, which showed that E.coli were mainly captured by the green barcodes; and the red and blue ones showed no obvious capture (Fig. S7). These results indicated that the aptamer-functionalized magnetic hydrogel barcodes can capture and distinguish bacteria specifically, And the colors were employed for
Fig. 4. (a) The relationship of the bacteria fluorescence intensities with the aptamer concentrations and capture time. (b) The relationship of the bacteria fluorescence intensity with the bacteria concentration. Error bars represent standard deviations.
1 mM. To optimize the capture time, the 1 mM AptE.coli decorated magnetic barcodes were incubated in same concentration of E.coli for different time (30, 60, 90, 120, 150, 180 and 210 min). It was found that the fluorescence intensity of the captured bacteria on the barcodes initially increased with incubation time, and 95% of capture could be achieved after a 2.5 h detection. Although a nearly complete capture could be achieved in 3–3.5 h, it wasted much time for the detection, so
Fig. 5. (a) Schematic diagram of the aptamer-decorated barcodes for capturing multiple types of bacteria. (b, c) Optical microscopy image and fluorescence image three kinds of barcodes after S.aureus capture. The red, green, and blue barcodes were decorated with AptS.aureus, AptE.coli, and no probes, respectively. Scale bar is 200 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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References
distinguishing different particles, which were immobilized with different aptamers respectively for the multiple bacteria detection. Thus, this barcodes technology can offer a new method to capture specific bacteria in the blood for bacteremia diagnosis and therapy.
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4. Conclusions In summary, we have demonstrated a new barcode technology that can simultaneously capture and detect multiple types of bacteria from blood sample. The barcodes are inverse opal structured magnetic PEG hydrogel particles. As the barcodes were encoded by their characteristic reflection peak, its code information remained stable during bacteria capture. The decorated aptamer probes in the PEG scaffolds specifically captured the bacteria, while the PEG hydrogel scaffold decreased the non-specificity adsorption of other bacteria. The tagged magnetic nanoparticles in the PEG scaffold imparted the barcodes with controllable movement under magnetic fields, which could not only increase the reaction speed, but also simplify the processing of the detection. It was demonstrated that the bacteria could be captured and identified even at low bacterial concentrations (100 CFU mL−1) within 2.5 h by the proposed magnetic hydrogel barcodes, which is effectively shortened in comparison with the “gold standard” in clinic. These results indicated the potential value of the barcodes for the clinical hematological infection diagnostics. Acknowledgements This work was supported by the National Science Foundation of China (Grant nos. 21473029 and 51522302), the NSAF Foundation of China (Grant no. U1530260), the Key Medical Projects of Jiangsu Province (Grant no. BL2014078), Key Discipline of Jiangsu Province (2016–2020), the National Science Foundation of Jiangsu (Grant no. BK20140028), and the Scientific Research Foundation of Southeast University. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2017.09.032.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Aptamer-based hydrogel barcodes for the capture and detection of multiple types of pathogenic bacteria ⁎
Yueshuang Xua, Huan Wangb, Chengxin Luana, Yuxiao Liub, Baoan Chena, , Yuanjin Zhaoa,b, a b
MARK
⁎⁎
Department of Hematology and Oncology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Inverse opal Barcode Bacteria Aptamer Magnetic properties
Rapid and sensitive diagnosing hematological infections based on the separation and detection of pathogenic bacteria in the patient's blood is a significant challenge. To address this, we herein present a new barcodes technology that can simultaneously capture and detect multiple types of pathogenic bacteria from a complex sample. The barcodes are poly (ethylene glycol) (PEG) hydrogel inverse opal particles with characteristic reflection peak codes that remain stable during bacteria capture on their surfaces. As the spherical surface of the particles has ordered porous nanostructure, the barcodes can provide not only more surface area for probe immobilization and reaction, but also a nanopatterned platform for highly efficient bioreactions. In addition, the PEG hydrogel scaffold could decrease the non-specificity adsorption by its anti-adhesive effect, and the decorated aptamer probes in the scaffolds could increase the sensitivity, reliability, and specificity of the bacteria capture and detection. Moreover, the tagged magnetic nanoparticles in the PEG scaffold could impart the barcodes with controllable movement under magnetic fields, which can be used to significantly increase the reaction speed and simplify the processing of the bioassays. Based on the describe barcodes, it was demonstrated that the bacteria could be captured and identified even at low bacterial concentrations (100 CFU mL−1) within 2.5 h, which is effectively shortened in comparison with the “gold standard” in clinic. These features make the barcodes ideal for capturing and detecting multiple bacteria from clinical samples for hematological infection diagnostics.
1. Introduction Bacteremia caused by bacterial bloodstream infections, such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and so on, can lead to vascular leakage, tissue damage, and multiorgan failure; these are associated with one-third of global mortality (Angus and van der Poll, 2013; Kailasa and Wu, 2012, 2013; Rocheteau et al., 2015). Identifying bacteria rapidly in the early stage of infection is significant to decrease high mortality (Yealy et al., 2014). However, current “gold standard” in clinical diagnosis of bacteremia usually needs 3–5 days of incubation and at least 12 h of growing on solid media to identify the bacteria (Sarkar et al., 2006). Various approaches have been devised to improve the sensitivity for bacterial identification, such as real-time polymerase chain reactions (Ottesen et al., 2006), fluorescent in situ hybridization, surface enhanced Raman scattering, and fluorescent probes (Kang et al., 2014). In spite of the improvement, these methods still require long-term blood culture and expensive equipment, which limit their widespread use in clinical applications. As an alternative,
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some simple cell capture platforms have been constructed to shorten the time needed for pathogen detection as well as identification. However, most of these methods were only carried out with single target test, and could not effectively distinguish between different bacteria simultaneously in a simple detection. Therefore, the development of a new platform that can capture and distinguish multiple types of bacteria simultaneously in a short time is highly desired. Barcodes(also called as encoded microcarriers), which encode information about their specific compositions and enable simple identification, have attracted increasing interest for multiple bioassays (Liu et al., 2014; Meng et al., 2015; Shi et al., 2013; Xu et al., 2017; Yang et al., 2008; Zhang et al., 2016; Zheng et al., 2014). Many kinds of encoding strategies have been proposed for the barcodes, including fluorescent molecules, quantum dots, photonic crystals, or graphical or shape-encoded microplates, and so on (Ge and Yin, 2008, 2011; Kanai et al., 2010; Lee et al., 2015; Mao et al., 2010; Shang et al., 2013; Sim et al., 2015; Song et al., 2015; Xu and Chen, 2015; Yu et al., 2009; Y.Q. Zhang et al., 2013, Y.S. Zhang et al., 2013; Zhao et al., 2014, 2015).
Corresponding author. Corresponding author at: Department of Hematology and Oncology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China. E-mail addresses: [email protected] (B. Chen), [email protected] (Y. Zhao).
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http://dx.doi.org/10.1016/j.bios.2017.09.032 Received 30 July 2017; Received in revised form 12 September 2017; Accepted 18 September 2017 Available online 20 September 2017 0956-5663/ © 2017 Elsevier B.V. All rights reserved.
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Scheme 1. Schematic diagram of the inverse opal structured magnetic hydrogel barcodes with aptamer probes for the bacteria capture.
hydroxy-2-methylpropiophenone (HMPP) photoinitiator were purchased from Sigma-Aldrich, Shanghai, China. Acrylic Acid (AA) was obtained from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Aladdin Reagent Co., Ltd., Shanghai, China. 2-Morpholinoethanesulfonic Acid (MES) was obtained from AMRESCO LLC, Solon, USA. Phosphate buffer saline (PBS, 0.05 M, pH 7.4) were self-prepared. All buffers were self-prepared using water purified in a Milli-Qsystem (Millipore, Bedford, USA). 30 healthy human blood samples were collected from the affiliated ZhongDa Hospital of Southeast University, Nanjing, China. All the collection and processing of human blood samples were carried out in accordance with the guidelines issued by the Ethical Committee of the Chinese Academy of Sciences.
Based on these barcodes, many distinct multiple assays have been carried out for high throughput biomolecule detection, gene function analysis, and clinical diagnosis (Fu et al., 2016). However, the encoded information of most barcodes would be confused or incomprehensible when their surfaces were covered by bacteria, and this could cause false decoding of the barcodes. In addition, the debatable specificity and reliability of their general surface morphology and biochemical modification, as well as the uncontrolled motion, have also limited the application of barcodes for the bacteria detection. Thus, the development of new barcodes-based bacteria capture and detection platform with distinct advantages is still required. Scheme 1 In this paper, we present a new aptamer-functionalized barcodes technology that can simultaneously capture and detect different types of pathogenic bacteria. The barcodes are poly (ethylene glycol) (PEG) hydrogel inverse opal particles with characteristic reflection peak codes that remain stable during bacteria capture on their surfaces (Choi et al., 2009; Hong et al., 2012; Shang et al., 2015; Stein et al., 2013; Y.Q. Zhang et al., 2013, Y.S. Zhang et al., 2013). The decorated aptamer probes in the PEG scaffolds could specifically capture the bacteria, while the PEG hydrogel scaffold could decrease the non-specificity adsorption of other targets. Due to the ordered porous nanostructure on the spherical surface, the barcodes can provide not only more surface area for probe immobilization and reaction, but also a nanopatterned platform for highly efficient bioreactions. In addition, the tagged magnetic nanoparticles in the PEG scaffold could impart the barcodes with controllable movement under magnetic fields, which can be used to significantly increase the reaction speed and simplify the processing of the bioassays. Thus, compared with other reported methods, our barcodes have such advangtages, include low non-specificity adsorption, good efficiency, fast capture speed and multiplex capture capacity. It will be demonstrated that the bacteria with low concentrations could be captured and identified very fast by the barcodes, which fully satisfied the clinical criteria during the hematological infection diagnostics.
2.2. Instruments The microfluidic device used for generating silica colloidal crystal beads was home-made. All reactions were finished in flat-bottom tubes and a constant temperature shaker (Thermomixer comfort 5355, Eppendorf, Germany). The microstructures of silica colloidal crystal beads and hydrogel photonic barcodes were characterized according to a scanning electron microscope (SEM, S-300N, Hitachi, Japan). Photographs of the two kinds of beads were taken by an optical microscope (BX51, Olympus, Japan) equipped with a CCD camera (MP5.0, Media Cybernetics Evolution). The reflectance spectra of the barcodes were recorded by the same microscope equipped with a fiber optic spectrometer (HR2000, Ocean Optics, USA). The fluorescence intensity was detected by a fluorescence microscope (BX53, Olympus, Japan). 2.3. Fabrication of inverse opal hydrogel magnetic barcodes Silica colloidal crystal beads (SCCBs) used as the template to fabricate this kind of inverse opal barcodes were fabricated using the microfluidic devices. The inverse opal barcodes were replicated from the voids of the template silica colloidal crystal beads. And the pre-gel solution used for the fabrication of inverse opal barcodes was composed of Poly (ethyleneglycol) diacrylate (PEG-DA) and Acrylic Acid (AA). Firstly, the dried silica colloidal crystal beads with different colors were immersed in pre-gel solution (20% PEG-DA, 10%AA and 1% HMPP) for 1 h. The liquid mixed solution could fill the gaps of silica colloidal crystal beads fully. Next, the mixture of beads and pre-gel solution was exposed to UV light for polymerizing the pre-gel solution in and out of the SCCBs. After polymerization, the hybrid beads of different colors could be extracted by stripping the pre-gel on the surface of the beads, and then remove the silica template with 4% hydrofluoric acid to obtain hydrogel inverse opal barcodes. Finally, the magnetic inverse opal barcodes could be obtained by saturating the barcodes with the magnetic nanoparticles.
2. Experimental section 2.1. Materials Six kinds of SiO2 nanoparticles with the size of 211, 260 and 307 nm were purchased from NanJing DongJian Biological Technology Co., Ltd. CY3 labeled rabbit polyclonal anti-human AFP antibody were purchased from Micro Biological Technology Company, Shanghai, China. Two kinds of aptamers (AptS.aureus and AptE.coli) were purchased from Ruibo Biological Technology Co., Ltd., Guangzhou, China. Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) were purchased from the inquiry network for microbial strains of China. FITC Concanavalin A was purchased from Sigma-Aldrich, Shanghai, China. Poly (ethyleneglycol) diacrylate (PEG-DA) with molecular weights of 700 and 2405
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Fig. 1. (a) The optical images of the monodisperse hydrogel barcodes; (b) reflection spectra and optical images of six kinds of hydrogel barcodes; (c) hydrogel barcodes; (d) hydrogel barcodes under a magnetic field. Scale bar is 200 µm.
reaction process, we use magnet to shuttle the barcodes in the samples constantly. Then wash away the residual infected blood around the barcodes twice with PBS buffer. The efficiency of detection is evaluated through fluorescence intensity of the barcodes. The method for detecting S.aureus is the same as that for E.coli. To validate the feasibility of applying magnetic hydrogel inverse opal barcodes in capturing multiple types of bacteria in blood, three kinds of magnetic inverse opal barcodes with characteristic reflection peaks at 600, 530, and 440 nm (referred to as red, green, and blue, respectively) were used in the experiment for multiple bacteria capture. For this purpose, the red inverse opal barcodes were coated with AptS.aureus, and the green inverse opal barcodes were with AptE.coli, while the blue one served as a control with no aptamer adsorption. These barcodes were then mixed and incubated in blood samples spiked with S.aureus or E.coli which were dyed with FITC Concanavalin A for 2.5 h at 37 °C with constant shuttle. Then wash away the residual infected blood around the barcodes twice with PBS buffer and observe the specificity of this experiment through fluorescence intensity of the barcodes. Subsequently, to image the samples by using a scanning electron microscope, the captured bacteria were fixed using 4% (v/v) paraformaldehyde overnight at 4 °C followed with dehydration with gradient ethanol gradually. The common concentration gradient of ethanol is from 20%, 40%, 60%, 80–100%, and the samples were dehydrated for over 20 min in each concentration.
2.4. Preparation of the aptamer-conjugated inverse opal hydrogel magnetic barcodes Two kinds of aptamer probes (AptS.aureus and AptE.coli) were dissolved in sterile water at the concentration of 10 mM. Firstly, treat the magnetic hydrogel inverse opal barcodes prepared before with 2Morpholinoethanesulfonic Acid (MES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for 30 min at 37 ℃ in the constant shaker. Then red inverse opal barcodes were coated with amino-modified AptS.aureus, and green barcodes were coated with amino-modified AptE.coli after being washed with PBS for 6 h at 25 ℃. Finally, the different functionalized inverse opal barcodes with own probes were prepared successfully after washing with buffer solution. 2.5. Bacterial sample culture The Luria−Bertani (LB) broth was prepared by dissolving 10 mg/ mL tryptone, 5 mg/mL yeast extract and 10 mg/mL Nacl in purity water followed with sterilization. The strains of S. aureus and E. coli were cultured with LB broth at 37 °C overnight, and the colony forming units (CFUs) were determined by measuring the optical density (OD) at 600 nm (OD600 = 1.0 is approximately 1.0 × 109 CFU mL−1). In detail, we first diluted the culture to obtain an appropriate OD value of about 1.0. After that, the bacterial solution with an OD600 value of about 1.0 was serially diluted and inoculated onto the solid medium to quantify the CFU mL−1.
3. Results and discussion 3.1. Design of the hydrogel barcodes
2.6. Selectivity Test of S. aureus and E.coli As a typical experiment, the inverse opal structured hydrogel barcodes were fabricated by replicating silica colloidal crystal bead templates. These bead templates were prepared by the ordered self-assembly of silica nanoparticles in microfluidic droplets during the evaporation of water. The ordered structure of the nanoparticles gives the colloidal crystal beads brilliant structural colors and reflection spectra (Fig. S1). To generate the hydrogel barcodes, the silica colloidal crystal bead templates were first immersed in a pre-gel solution. After the pre-gel solution had filled the voids between the nanoparticles of
First of all, we dyed the bacteria with FITC Concanavalin A under protection from light for half an hour. Then, the bacteria were washed three times with PBS buffer by centrifugation to remove free FITC Concanavalin A. In a typical experiment, human blood was spiked with E.coli to simulate bacteremia at a final concentration of 104 CFU mL−1. Subsequently, almost ten aptamer-conjugated magnetic inverse opal barcodes were added to 100 μL infected blood, and the mixture was incubated for 2.5 h followed by magnetic separation. In this whole 406
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Fig. 2. SEM images: (a, b) surface structure of the colloidal crystal beads, which indicated a commendable hexagonal close packing structure; (b) inverse opal barcodes with an interconnected porous surface; (c) the captured E.coli on the surface of the AptE.coli-decorated inverse opal hydrogel barcodes; (d) the captured S.aureus on the surface of the AptS.aureus-decorated inverse opal hydrogel barcodes. Scale bars are 500 nm in (a-b), 2 µm in (c) and 500 nm in (d).
morphology on their surfaces (Fig. 2d). It was worth to mention that when the bacteria were captured on the barcodes, they did not affect the ordered structure of the whole sphere. Thus, the encoded elements of the inverse opal barcodes remained constant during the bacteria capture. Due to the ordered porous nanostructure, the hydrogel barcodes were imparted with a photonic band gap (PBG) property and show the corresponding structural color or characteristic reflection peak. Under normal incidence, the peak positions λ of the barcodes can be estimated by Bragg's equation [Eq. (1)]
the templates by the capillary force, the pre-gel solution in and out of the bead templates was polymerized by using ultraviolet light. Finally, the inverse opal structured hydrogel barcodes were obtained by removing the pre-gel on the surface of the templates and etching the silica nanoparticles in the templates with hydrofluoric acid (Fig. 1). To impart the resultant barcodes additional feature, an ultrafine magnetic nanoparticles solution was employed for immersing the inverse opal structured hydrogel barcodes. This treatment imparted the barcodes with controllable movement under magnetic fields (Fig. 1c, S1c-d), which was potential to increase the reaction speed and simplify the processing of the bioassays. The microstructures of the inverse opal structured magnetic hydrogel barcodes and their templates were observed with a scanning electron microscope (SEM). It can be seen from Fig. 2 that the silica nanoparticles on the surface of the template beads mainly formed a hexagonal alignment (Fig. 2a). Thus, the inverse opal barcodes replicated from the templates should have a similar highly ordered three dimensional (3D) inverse opal structure and hexagonal symmetrical porous surface. As expected, it can be observed that the barcodes had an interconnected and hexagonal symmetrical porous surface (Fig. 2b), which would provide a nanopatterned platform for highly efficient entrapping bacteria. To capture bacteria, different types of probes could be employed. Among these probes, aptamers are single stranded DNA or RNA oligonucleotides that can bind to variety of targets with high selectivity and strong affinity. Compared to other biomolecules, aptamers have some advantages, including small size, easy modification, and synthesis. Recently, several bacterial aptamers with high selectivity have been developed, so they are ideal options to detect specific bacteria from blood samples. As a proof of concept in this work, we used AptS.aureus or AptE.coli (Fig. S2, and the aptamer sequences were in Table S1) to capture S.aureus or E.coli, respectively. To confirm their ability, the AptE.coli-decorated barcodes were incubated in E.coli suspension and observed under SEM after the reaction. Fig. 2c showed that large numbers of E.coli were captured on the surface of the inverse opal barcodes, and the normal morphology of E.coli was maintained. Similarly, the AptS.aureus-decorated barcodes captured several S.aureus with normal
λ = 1.633dnaverage
(1)
where d is the center-to-center distance between two neighboring nanopores, and naverage is the average refractive index of the barcodes. Therefore, by using different sizes of silica nanoparticles assembled templates, a series of hydrogel barcodes with different sizes of nanopores and reflection peaks could be obtained for the coding (Fig. 1b). As there was no dye or other materials related with the code, concerns about chemical instability and the fluorescence background during the bacteria capture and detection were not necessary. 3.2. Optimization of the material To reduce the nonspecific adsorption of the proteins and other cells to the barcodes during the bacteria capture and detection, the PEG hydrogel, which has the advantages of anti-adhesive effect and mechanical strength, was employed as the scaffold material of the barcodes. In addition, in order to immobilize the amino-modified aptamers on the surface of the barcodes, the acrylic acid with a large number of carboxyl was also used as the hydrogel component. Thus, the pre-gel solution consisted of the poly(ethylene glycol) diacrylate (PEG-DA), acrylic acid and 2-hydroxy-2-methylpropiophenone photoinitiator. In general, lower concentration of PEG-DA could offer larger grid to enhance the actives of the immobilized aptamer probes. To confirm this, five kinds of inverse opal barcodes with different concentration of PEGDA were fabricated and immobilized with probes (Fig. S3). Then these barcodes were incubated in the fluorescence labeled targets. It was 407
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Fig. 3. Laser scanning confocal microscopy (LSCM) images of the captured E.coli bacteria by different hydrogel barcodes: (a) hydrogel barcodes without AptE.coli probes; (b) hydrogel barcodes with AptE.coli probes; (c) magnetic hydrogel barcodes with AptE.coli probes. From left to right: cross-section fluorescent images, merged cross-section fluorescent and white-light images, and 3D fluorescent images. Scale bar is 50 µm.
found that the fluorescence intensity of the barcodes with lowest PEGDA concentration (20%) was higher than those with 40%, 60% and 80% PEG-DA concentrations (Fig. S4), this indicated much targets were captured. Although a much higher capture performance would be achieved when the concentration of PEG-DA was lower than 20%, the barcodes showed poor mechanical properties. Thus 20% PEG-DA was used to fabricate all the hydrogel barcodes for the following experiments. It was worth to mention that the fluorescence intensity was pretty low for the control barcodes group that without aptamer probes immobilization, which confirmed the anti-adhesive effect of the PEG hydrogel material.
without AptE.coli probes, hydrogel barcodes with AptE.coli probes, and magnetic hydrogel barcodes with AptE.coli probes, respectively (Fig. 3). It can be seen that the latter two kinds of barcodes with aptamer probes can capture more bacteria, which means that the aptamer probes with strong affinity can surely increase the capture specificity. In addition, because the magnetic barcodes was shuttled in the samples constantly with the controllable movement under magnetic fields (Fig. 1c, Fig. S5), they could capture more bacteria on their surfaces than those barcodes without controllable movement, as shown in Fig. 3b and c. To further increase the capture efficiency of the bacteria on the magnetic hydrogel barcodes, we have also optimized the concentrations of the aptamer probes and the incubation times during the capture (Fig. 4a). In these processes, the barcodes were decorated with different concentrations of AptE.coli (50, 100, 200, 500, 1000and 2000 μM) for the probes immobilization, and then these barcodes were added into the same concentration of E.coli for the same capture time. It was found that with the increasing of the concentration of the aptamer, the fluorescence intensity of the captured bacteria on the barcodes gradually increased, and they saturated when the concentration exceeded
3.3. Optimization of the reaction conditions To visually observe the captured bacteria on the surface of the barcodes, E.coli were stained by using FITC Concanavalin A for 30 min at room temperature. After this staining, a laser scanning confocal microscopy (LSCM) was employed for the cross-section and three-dimensional images of the E.coli on the surface of the hydrogel barcodes 408
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we chose to use the capture time of 3.5 h to ensure nearly complete capture in this work for faster detection. This capture time indicated a much faster detection process than the previous method in clinic, and this advantage may realize the rapid diagnosis of bacteria in future.
3.4. Bacteria capture For the optimized experiments mentioned above, the bacteria were designed with high concentrations in the blood samples for the researches. However, the infectious dose of bacteria in the real blood sample was usually very low. Thus, to evaluate the capture sensitivity of the magnetic hydrogel barcodes at low bacterial concentrations, human blood samples were also spiked with different concentrations of E.coli (100, 500, 1000, 2500, 5000, and 104 CFU mL−1) for the barcodes detection. The results showed that the numbers and the relative fluorescence intensities of E.coli on the barcodes increased with the increasing of the concentration of the E.coli in the blood samples (Fig. 4b and Fig. S6). The fluorescence intensity of the background was 17, and the standard deviation was 10, so it is noteworthy that the bacteria could be detected even at a low concentration of 100 CFU mL−1; this indicated the feasibility of the magnetic hydrogel barcodes for bacteria detection in clinical blood samples. To demonstrate the specificity of different aptamers and the capability of the magnetic hydrogel barcodes for capturing multiple types of bacteria in blood, three kinds of magnetic barcodes with characteristic reflection peaks at 600, 530, and 440 nm (referred to as red, green, and blue, respectively) were used for the test, as shown in Fig. 5. In this experiment, the red and green barcodes were decorated with AptS.aureus and AptE.coli, respectively; while the blue one served as a control without aptamer probe immobilization. These barcodes were then mixed together and incubated in healthy human blood spiked with low concentration of S.aureus. It was found that S. aureus were mainly captured on the red barcodes due to the specific binding between the S.aureus and the AptS.aureus, while the green and blue barcodes showed no obvious capture (Fig. 5c). By using E.coli to instead S.aureus in the blood, it could got an inverse result, which showed that E.coli were mainly captured by the green barcodes; and the red and blue ones showed no obvious capture (Fig. S7). These results indicated that the aptamer-functionalized magnetic hydrogel barcodes can capture and distinguish bacteria specifically, And the colors were employed for
Fig. 4. (a) The relationship of the bacteria fluorescence intensities with the aptamer concentrations and capture time. (b) The relationship of the bacteria fluorescence intensity with the bacteria concentration. Error bars represent standard deviations.
1 mM. To optimize the capture time, the 1 mM AptE.coli decorated magnetic barcodes were incubated in same concentration of E.coli for different time (30, 60, 90, 120, 150, 180 and 210 min). It was found that the fluorescence intensity of the captured bacteria on the barcodes initially increased with incubation time, and 95% of capture could be achieved after a 2.5 h detection. Although a nearly complete capture could be achieved in 3–3.5 h, it wasted much time for the detection, so
Fig. 5. (a) Schematic diagram of the aptamer-decorated barcodes for capturing multiple types of bacteria. (b, c) Optical microscopy image and fluorescence image three kinds of barcodes after S.aureus capture. The red, green, and blue barcodes were decorated with AptS.aureus, AptE.coli, and no probes, respectively. Scale bar is 200 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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References
distinguishing different particles, which were immobilized with different aptamers respectively for the multiple bacteria detection. Thus, this barcodes technology can offer a new method to capture specific bacteria in the blood for bacteremia diagnosis and therapy.
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4. Conclusions In summary, we have demonstrated a new barcode technology that can simultaneously capture and detect multiple types of bacteria from blood sample. The barcodes are inverse opal structured magnetic PEG hydrogel particles. As the barcodes were encoded by their characteristic reflection peak, its code information remained stable during bacteria capture. The decorated aptamer probes in the PEG scaffolds specifically captured the bacteria, while the PEG hydrogel scaffold decreased the non-specificity adsorption of other bacteria. The tagged magnetic nanoparticles in the PEG scaffold imparted the barcodes with controllable movement under magnetic fields, which could not only increase the reaction speed, but also simplify the processing of the detection. It was demonstrated that the bacteria could be captured and identified even at low bacterial concentrations (100 CFU mL−1) within 2.5 h by the proposed magnetic hydrogel barcodes, which is effectively shortened in comparison with the “gold standard” in clinic. These results indicated the potential value of the barcodes for the clinical hematological infection diagnostics. Acknowledgements This work was supported by the National Science Foundation of China (Grant nos. 21473029 and 51522302), the NSAF Foundation of China (Grant no. U1530260), the Key Medical Projects of Jiangsu Province (Grant no. BL2014078), Key Discipline of Jiangsu Province (2016–2020), the National Science Foundation of Jiangsu (Grant no. BK20140028), and the Scientific Research Foundation of Southeast University. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2017.09.032.
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