Magnetism-Resolved Separation and Fluorescence Quantification for

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Magnetism-resolved separation and fluorescence quantification for near-simultaneous detection of multiple pathogens Linyao Li, Qingjing Li, Ziyi Liao, Yan Sun, Quansheng Cheng, Yang Song, Erqun Song, and Weihong Tan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02572 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 14, 2018

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Analytical Chemistry

Magnetism-resolved separation and fluorescence quantification for near-simultaneous detection of multiple pathogens

Linyao Li, † Qingjin Li, † Ziyi Liao, † Yan Sun, † Quansheng Cheng, † Yang Song, † Erqun Song†,*, Weihong Tan‡, §

†

Key Laboratory of Luminescence and Real-Time Analytical Chemistry, Ministry of Education,

College of Pharmaceutical Sciences, Southwest University, Chongqing, 400715, People’s Republic of China. Fax: +862368251225; Tel: +862368251225. E-mail: [email protected] ‡

Molecular Science and Biomedicine Laboratory, State Key Laboratory for Chemo/Bio-Sensing

and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, and Collaborative Research Center of Molecular Engineering for Theranostics, Hunan University, Changsha 410082, People's Republic of China. §

Center for Research at Bio/Nano Interface, Department of Chemistry and Department of

Physiology and Functional Genomics, Health Cancer Center, UF Genetics Institute and McKnight Brain Institute, University of Florida, Gainesville, Florida 32611-7200, United States

ABSTRACT

In the modern era of molecular evidence-based medicine and advanced biomedical technologies, the rapid, sensitive and specific assay of multiple pathogens is critical to, but largely absent from, clinical practice. Therefore, to improve the current ordinary separation and collection method, we report herein a strategy of magnetism-resolved 1

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separation and fluorescence quantification for near-simultaneous detection of multiple pathogens, followed by the direct antimicrobial susceptibility testing (AST). To accomplish this strategy, we utilized aptamer-modified fluorescent-magnetic multifunctional nanoprobes (apt-FMNPs). FMNPs with intriguing different magnetic responses and excellent fluorescence quality were first self-assembled based on metal coordination interaction using (3-mercaptopropyl) trimethoxysilane, magnetic γ-Fe2O3, and fluorescent quantum dots as matrix components. Then, aptamers, which specific to target pathogens of Escherichia coli O157:H7 (E. coli) and Salmonella typhimurium (S. typ), were conjugated with FMNPs to yield apt-FMNPs nanoprobes for multiple pathogens assay. Based on the discrepant magnetic response of pathogen@nanoprobes complex under the identical external magnetic field, the model bacteria were fished out by magnetic adsorption at different time points and subjected to fluorescence quantification with good linear ranges and detection limits within 1h. Multiple pathogens spiked in real samples were also effectively detected by the apt-FMNPs and sequentially fished out for AST assay, which showed similar results to that for pure pathogens. The apt-FMNPs-based strategy of near-simultaneous detection of multiple pathogens shows promise for the potential application in the diagnosis and treatment of pathogen-related infectious diseases.

Key words: magnetism, fluorescence, pathogen, multifunctional nanoprobes, assembly

2


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INTRODUCTION Multiple pathogens may coexist in one sample,1 usually with low infectious

dosages (ID).2 This calls for the development of a rapid, sensitive and specific analytic method for simultaneously or near-simultaneously detection of multiple pathogens. Conventional culture/colony counting approaches for identifying pathogens are slow and suffer from the lack of specificity and sensitivity. 3, 4 In recent years, various new methods have been extensively explored to rapidly and simultaneously detect multiple pathogens, including polymerase chain reaction (PCR) techniques,5-7 immunoassays8-12 and other detection techniques,13-16. However, some of these methods have non-ignorable drawbacks, such as poor reproducibility, false-positive results and high cost.17, 18 Whilst other strategies cannot simultaneously separate and collect different types of pure bacterium. Therefore, these steps need to be addressed in the context of downstream studies, in particular those involving antimicrobial susceptibility testing (AST).19, 20 Recently, fluorescent magnetic multifunctional nanoparticles (FMNPs) have been reported to successfully separated and detected multiple proteins,21 viruses,22 cells23 and DNA sequences24 based on a multi-optical-labeled analytic strategy. However, the inherent spectral overlap interference of fluorescent-encoded FMNPs has limited their applications. From another perspective, they could not separate single component from a multicomponent complex for further analysis.21-24 To improve the strategy for multiple-component assay, we have recently fabricated a set of magnetic-encoded FMNPs with variable magnetic response, along with good 3



fluorescence properties through microemulsion25 and electrostatic interactions-based self-assembly26 by employing γ-Fe2O3 nanoparticles and quantum dots (QDs) as magnetic and fluorescent components, respectively. However, for the former strategy, the number of embedded γ-Fe2O3 and QDs is constant and limited due to the constant internal space of magnetic-encoded FMNPs once their size was fixed, which results in restricted fluorescent and magnetic properties of FMNPs. Envisioning that, when trying to expand the magnetic response of the FMNP by increasing the number of γ-Fe2O3, their fluorescent intensity of FMNP will reduce due to the decrease of the number of embedded QDs, vice versa. Moreover, as the encapsulated γ-Fe2O3 and QDs touch each other directly in the interior of silicon nanospheres, the fluorescence intensity of QDs is directly badly affected by γ-Fe2O3.27 And for magnetic-encoded FMNPs obtained by the latter strategy, they are susceptible to their surroundings, and their size is inhomogeneous owing to layer-by-layer assembly. In this study, we have advanced our previous research and successfully fabricated novel magnetic-encoded FMNPs with good fluorescence properties, differential magnetic potential, and near-uniform size using a simple and convenient metal coordination-based self-assembly method, as shown in Scheme 1A. Nucleic acids aptamers have advantages, for instance, high affinity and specificity towards their targets, stability and cost-effectiveness compared to antibodies.28-31 Thus, we combined aptamers with magnetic-encoded FMNPs to obtain apt-FMNPs nanoprobes (Scheme 1B) for the rapid, sensitive and near-simultaneous detection and separation of multiple pathogenic bacterial strains for downstream study (Scheme 2). Here, Escherichia coli O157:H7 4


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(E. coli) and Salmonella typhimurium (S. typ) were employed as model pathogenic bacteria since they are the most common causes of foodborne illness, such as diarrhea, hemolytic uremic syndrome and salmonellosis.3, 32 The study showed that E. coli and S. typ could be separated in two minutes and detected with good linear range and detection limit by using apt-FMNPs nanoprobes with differential magnetism and good fluorescence intensity. Furthermore, the proposed method showed good feasibility for assay of the two target bacterial strains in real samples, such as milk, human serum, and human urine. Finally, the two pathogenic bacterial strains were sequentially and

Scheme 1 (A) Schematic of fabricating magnetic-encoded FMNPs based on the strong thiol-metal coordination between MPS nanosphere and γ-Fe2O3 or CdSe/ZnS QDs nanoparticles. FMNPs with different degrees of magnetism controlled by simply adjusting the concentration of γ-Fe2O3 nanoparticles initially added; (B) Bio-functionalization of FMNPs with aptamers. (MPS: (3-mercaptopropyl) trimethoxysilane; W-FMNPs: fluorescent magnetic multifunctional nanoparticles (FMNPs) with weak magnetic potential; S-FMNPs: FMNPs with strong magnetic potential; APTES: (3-aminopropyl) triethoxysilane). 5



magnetically collected from human urine sample by the apt-FMNPs nanoprobes and directly subjected to AST assay, which showed similar results to that from pure two pathogens. The rapid recognition of multiple pathogens, followed by direct AST assay, demonstrated the potential applicability of this strategy in diagnosis of bacterial infectious disease, and potential aid in reasonable administration by speeding up the AST assay.

Scheme 2 Schematic of sequential magnetic separation of target bacteria captured by apt-FMNPs from the mixture under an external magnetic field, followed by fluorescence analysis and AST assay.

EXPERIMENTAL SECTION Materials and Apparatus. Iron (III) acetylacetonate, (3-mercaptopropyl)

trimethoxysilane (MPS), ethyl silicate (TEOS), and (3-aminopropyl) triethoxysilane (APTES) were purchased from Aladdin Industrial Inc. (Shanghai, China). All antimicrobial agents, including Tetracycline (Tet), Ampicillin (Amp), Gentamicin (Gen), and Nalidixic acid (Nal) were purchased from Hangzhou Microbial Reagent Co. Ltd. (China). Oil-soluble CdSe/ZnS quantum dots (QDs) were purchased from Wuhan Jiayuan Quantum Dots Co. Ltd. (China). E. coli (CICC 21530) was obtained 6


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from the China Center of Industrial Culture Collection. S. typ (ATCC 14028), Staphylococcus aureus (S. aureus, ATCC 29213), and Listeria monocytogenes (L. monocytogenes ATCC 19115）were obtained from China General Microbiological Culture Collection Center. The aptamers, random sequence DNA (rDNA), and the primers for PCR (in Table S1 and S3 in the Supporting Information) were synthesized by Shanghai Sangon Biological Science & Technology Company (Shanghai, China). The fluorescence spectra were obtained using a fluorescence spectrophotometer (F-7000, Hitachi). Morphology and microscopic structure were characterized using a transmission electron microscope (TEM) (JEM 1200EX, JEOL Ltd.) and scanning electron microscope (SEM) (JSM-7500F, JEOL Ltd.). The magnetism of γ-Fe2O3 nanoparticles and W/S-FMNPs were measured by a vibrating sample magnetometer (VSM) (LDJ-9600, MicroSense Corporation). Zeta potential and size distribution were measured using a Malvern Zetasizer Nano ZS ZEN3600 instrument (Malvern Instruments, United Kingdom). Fluorescent images were obtained under an inverted fluorescence microscope (Olympus IX71). A TB246 magnetic scaffold was used to achieve the magnetic separation and capture. (Promega, Beijing Biotech Co. Ltd.).

Bacteria Culture and Counting. All bacteria were grown in a liquid culture. S. typ, E. coli and S. aureus were grown in beef extract peptone medium, and L. monocytogenes was grown in peptone-yeast extract-glucose medium broth at 37 ºC with continuous shaking. The colonies on the plates were counted to determine the number of colony-forming units per milliliter (cfu/mL) by the conventional agar plate-counting method after bacteria revived at 37 ºC for 18 h. 7



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Assembly of FMNPs with Different Degrees of Magnetism. MPS nanospheres and γ-Fe2O3 nanoparticles were first prepared according the previous reports33,

34

and their characterizations were showed in Figure S1. Then the

magnetic-encoded FMNPs were fabricated using layer-by-layer assembly,35,

36

as

illustrated in Scheme 1. Typically, γ-Fe2O3 nanoparticles were first assembled on the surface of MPS nanospheres (5 mg/mL) by simply mixing them in 2 mL cyclohexane under vigorous stirring for 1 h, producing MPS@γ-Fe2O3 nanocomposites. Before another MPS layer coated, the MPS@γ-Fe2O3 nanocomposites were inversed phase from oil phase to water phase after mixed with CTAB (10 mg/mL, 5 mL) and sonicated for 10 min, following heating to 80ºC for 30 min to evaporate the remaining trace amount of cyclohexane. After sequentially adding ammonia (1 mL) and MPS precursor (80 µL) to the above solution and stirring for 3 h, the nanocomposites were collected by magnetic separation and washed with anhydrous ethanol. Oil phase CdSe/ZnS QDs (3 mg/mL, 500 µL) were then assembled on the surface of nanocomposites (10 mg/mL, 1 mL) by mixing them in n-hexane solution with shaking for 1 h to obtain MPS@γ-Fe2O3@QDs nanocomposites. The final products of MPS@γ-Fe2O3@QDs@SiO2 (FMNPs) were fabricated by silanization with TEOS. For silicon capping with TEOS, MPS@γ-Fe2O3@QDs dispersed in anhydrous ethanol (1 mg/mL, 5 mL) were mixed with ammonia (800 µL) under vigorous magnetic stirring, and then TEOS (200 µL) was immediately injected into the solution every 20 min until the total volume of TEOS reached 600 µL with continuous stirring. After reaction for another 20 min, the products of MPS@γ-Fe2O3@QDs@SiO2 (FMNPs) 8


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were collected by magnetic separation and washed with anhydrous ethanol. The multi-magnetic FMNPs with weak and strong magnetic potentials, abbreviated as W-FMNPs and S-FMNPs, respectively, could be obtained by adjusting the concentration of γ-Fe2O3 nanoparticles initially added. The final concentrations of γ-Fe2O3 used for assembly W-FMNPs and S-FMNPs are 0.5 mg/mL and 5 mg/mL, respectively. Preparation of Aptamer-Modified FMNPs (apt-FMNPs) Nanoprobes. First, magnetic-encoded FMNPs were modified with an amino group by using APTES for further modification according to the published strategy.25 Briefly, FMNPs (0.5 mg/mL) were dispersed in a mixture solution containing 20 mL of anhydrous ethanol and 100 µL of ammonia and stirred for 10 min. Then, 100 µL of APTES were added dropwise to the above solution and reacted at room temperature for 6 h under continuous stirring, producing amino group-modified FMNPs (NH2-FMNPs). Then, NH2-FMNPs (1 mg/mL) were functionalized with avidin (1 mg/ mL) which were first activated by incubation with EDC (1.0 mg) and NHS (2.5 mg) for 15 min under gentle shaking in PBS (pH 7.4, 0.01 M, 1 mL)37. After 3 h incubation and gentle shaking at room temperature, the prepared avidin-conjugated FMNPs (avidin-FMNPs) were washed with PBS and resuspended in PBS. Finally, apt-FMNPs, or rDNA-modified FMNPs, were prepared by incubating avidin-FMNPs (1 mg/mL, 1 mL) with biotin-labelled aptamer or biotin-labelled random DNA (5 µM, 200 µL) at 37 ºC with shaking for 40 min. Quantitative Detection of S. typ and E. coli. Specifically, 100 µL of various 9



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concentrations 0, 10, 101.2, 101.4, 101.6, 101.8, 102, 103, 104, 105, 106, 107 and 108 cfu/mL of each target pathogens (S. typ and E. coli) were incubated with its corresponding apt-FMNPs nanoprobe (5 mg/mL, 50 µL), respectively, for 40 min at 37 ºC in 350 µL of SELEX buffer. Then the pathogen@nanoprobes complexes were magnetically separated and resuspended in 500 µL of SELEX buffer for fluorescence spectrum measurements (recorded at λex/em = 370/605 nm). Near-Simultaneous Assay of Multiple Pathogenic Bacteria in Authentic Samples by Apt-FMNPs Nanoprobes. The qualified milk samples were purchased from a local supermarket, and human serum and human urine were supplied by the Department of Oncology, the Ninth People's Hospital of Chongqing (China) from healthy volunteers. The three authentic samples, spiked with S. typ and E. coli at known concentration, together with S. aureus and L. monocytogenes (each 108 cfu/mL), were analyzed with apt-FMNPs nanoprobes to evaluate the applicability of the proposed strategy for downstream assay of target bacteria in authentic samples, as treated in the above procedures.

RESULTS AND DISCUSSION Fabrication and Structural Characterization of FMNPs. In this study, the

FMNPs

(MPS@γ-Fe2O3@QDs@SiO2)

nanocomposites

were

fabricated

by

self-assembling MPS nanospheres with magnetic γ-Fe2O3 nanoparticles/fluorescent QDs layer-by-layer (LBL) based on thiol-metal coordination followed by silanization, and two degrees of magnetic potential, i.e., W(eak)-FMNPs and S(trong)-FMNPs, could be obtained by adjusting the amount of γ-Fe2O3 upon assembly of the initial 10


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layer as shown in Scheme 1A. LBL assembly is an effective method for creating nanostructures from separate components owing to its simplicity and self-limiting nature, and silanization provides a nontoxic silica shell, making the whole nanoparticle relatively stable and conducive to further applications.25,

35

MPS

nanospheres and γ-Fe2O3 nanoparticles were first prepared following a reported strategy, then γ-Fe2O3 nanoparticles characterized by TEM, X-ray diffraction (XRD, X'Pert3 Powder, PANalytical B.V.), and VSM (Figure S1). The γ-Fe2O3 nanoparticles were directly attached to the surface of MPS nanospheres by simply stirring the mixture of the two components based on the strong interaction between thiol from MPS and iron from γ-Fe2O3 to get MPS@γ-Fe2O3 with weak magnetic potential (W-MPS@γ-Fe2O3) and strong magnetic potential (S-MPS@γ-Fe2O3). Figure 1A (W-MPS@γ-Fe2O3) and D (S-MPS@γ-Fe2O3) show the smaller γ-Fe2O3 nanoparticles assembled on the surface of larger MPS nanospheres and also an apparent difference in

the

number

of

γ-Fe2O3

for

products

of

MPS@γ-Fe2O3.

Then,

the

MPS@γ-Fe2O3@QDs nanocomposites with weak or strong magnetic potential (W-MPS@γ-Fe2O3@QDs, S-MPS@γ-Fe2O3@QDs) were obtained by assembling fluorescent QDs based on a principle similar to that for assembly of γ-Fe2O3 after subjecting MPS@γ-Fe2O3 to sulfhydration with MPS precursor. As shown in Figure 1B (W-MPS@γ-Fe2O3@QDs) and E (S-MPS@γ-Fe2O3@QDs), many smaller QDs were assembled, making a rough surface for the products of MPS@γ-Fe2O3@QDs. Finally, the products of MPS@γ-Fe2O3@QDs were encapsulated in a silica shell by silanization with ethyl silicate precursor to obtain the end product of 11



MPS@γ-Fe2O3@QDs@SiO2 with different loads of the magnetic component (Figure 1C: W-FMNPs and F: S-FMNPs). Compared with the products imaged in Figure 1B and E, the end products show a relatively smooth surface with size of about 200 nm. Considering the TEM results, it could be concluded that the FMNPs with different magnetic nanoparticle loading had been fabricated as expected. The hydrodynamic size distribution of products at each self-assembly procedure was shown in Figure S2. According to the specific calculating methods38 which were presented in supporting information, the approximate average numbers of γ-Fe2O3 in one single W-FMNP and S-FMNP are about 20 and 108 respectively, while the numbers of QDs in one single W-FMNP and S-FMNP are about 264 and 322, respectively (Table S2).

Figure 1. TEM images of the procedure of self-assembly of hydrophobic nanoparticles on MPS nanospheres. (A) W-MPS@γ-Fe2O3, (B) W-MPS@γ-Fe2O3@QDs, (C) W-MPS@γ-Fe2O3@QDs@SiO2 (W-FMNPs). (D) S-MPS@γ-Fe2O3, (E) S-MPS@γ-Fe2O3@QDs, (F) S-MPS@γ-Fe2O3@QDs@SiO2 (S-FMNPs). Characterization of FMNPs and apt-FMNPs. As shown in Figure S3A, the fluorescence intensity of S-FMNPs was weaker than that of W-FMNPs. Based on published work,27 this difference could be ascribed to the difference in the number of γ-Fe2O3 nanoparticles in S-FMNPs, which was more than that in W-FMNPs. The 12


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magnetic properties of the FMNPs were investigated using a VSM at room temperature. According to magnetization curves for W/S-FMNPs shown in Figure S3B, the W/S-FMNPs were all super-paramagnetic, with saturation magnetizations of 5.6 emu/g and 16.4 emu/g, respectively. The difference in saturation magnetization could be attributed to the different assembly amount of γ-Fe2O3 in the FMNPs. The above results implied that W/S-FMNPs could be separated from one another under an external magnetic field, which was proved by the different magnetic response of FMNPs to the external magnetic field generated by a magnetic scaffold (Figure S3C). As shown in Figure S3C, the two sets of S-FMNPs and W-FMNPs could be collected on the wall of the tube within 60 s and 120 s, respectively, demonstrating the sequential separation ability of W/S-FMNPs based on collection time in a given magnetic field. To further verify their sequential separation ability, W-FMNPs and S-FMNPs fabricated by using QDs with red (605 nm) and green (525 nm) fluorescence colour respective were mixed together and then subjected to magnetic separation, which was demonstrated by fluorescence microscopy images. It could be found that, there are many green fluorescent dots and red fluorescent dots in the fluorescence microscopy image in Figure S3D. And for the collection (Figure S3E) and suspension (Figure S3F) at 60 s magnetic separation for the W/S-FMNPs mixture, only one major fluorescence colour of fluorescent dots (605 nm or 525 nm) was observed. The effects of different buffer solutions and storage times on the fluorescence

13



intensity and magnetic response properties of the W/S-FMNPs were investigated, and the results show good stability of the FMNPs, as shown in Figure S4. Then the FMNPs were bio-functionalized with aptamers to obtain apt-FMNPs nanoprobes after the specific recognition of aptamers to their corresponding target bacteria was validated (Figure S5). The negative shift zeta potential value (Figure S6A, C) of apt-FMNPs compared to their precursors and the dual fluorescence emission peaks (Figure S6B, D) of apt-FMNPs demonstrated the successful preparation of apt-FMNPs nanoprobes. Then the amount of aptamer used for coupling with FMNPs was optimized by measuring the fluorescence intensity of supernatant after the apt-FMNPs nanoprobes removed using a magnet. According to Figure S7, an amount of 5 µM and 200 µL for aptamer was used in this study. Specific Recognition of Pathogenic Bacteria using Apt-FMNPs Nanoprobes. First, the specificity of apt-FMNPs nanoprobes was verified through fluorescence microscopy imaging based on the colocalization of double fluorescent signals from dye-aptamer and apt-FMNPs nanoprobes on the surface of bacteria. As shown in Figure S8, Cy3-apt-E. coli-W-FMNPs and FAM-apt-S. typ-S-FMNPs had affinity to their target bacteria of E. coli and S. typ, respectively, while they showed no affinity to nontarget bacteria (S. aureus and L. monocytogenes). In addition, Cy3-rDNA-W-FMNPs, FAM-rDNA-S-FMNPs and naked S/W-FMNPs had no affinity to the target S. typ and E. coli, respectively. Moreover, the specific bindings of the probes to target bacteria were further confirmed by SEM imaging. SEM images in Figure 2A and 2B showed that S. typ and E. coli could efficiently be recognized by 14


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apt-E. coli-W-FMNPs and apt-S. typ-S-FMNPs respectively, and the nontarget bacteria (L. monocytogenes and S. aureus) could not be recognized when respectively incubated with the mixture of apt-E. coli-W-FMNP and apt-S. typ-S-FMNPs as shown in Figure 2E and 2F. Figure 2C and 2D showed rDNA-W-FMNPs and rDNA-S-FMNPs could not combine with S. typ and E. coli respectively, which is consistent with the results in Figure S8.

Figure 2. SEM images of E. coli after incubated with apt-E. coli-W-FMNP (A) and rDNA-W-FMNP (C), S. typ after incubated with apt-S. typ-S-FMNPs (B) and rDNA-S-FMNPs (D), L. monocytogenes (E) and S. aureus (F) after incubated with the mixture of apt-E. coli-W-FMNP and apt-S. typ-S-FMNPs, respectively. Next, the recognition, capture and separation ability of apt-FMNPs nanoprobes for their corresponding targets were further studied by mixing the two types of apt-S/W-FMNPs nanoprobes with a mixture of S. typ and E. coli. After the whole mixture was subjected to incubation and sequential magnetic separation based on their different magnetic separation times, as illustrated in Scheme 2, the captures were imaged under fluorescence microscopy. In the dual mixture of S. typ and E. coli after incubating with Cy3-apt-E. coli-W-FMNP and FAM-apt-S. typ-S-FMNPs before magnetic separation, Figure 3A showed the simultaneous appearance of three fluorescence emission peaks of FAM, Cy3 and QDs at 520, 560, and 605 nm excited 15



by 490 nm. When the same dual mixture imaged under a fluorescence microscope (Figure 3B), there are some orange fluorescence dots (from Cy3) under excited with green light (Figure 3B, b) while there are both green fluorescence dots (from FAM) and red fluorescence dots (from QDs in FMNPs) under excited with blue light (Figure 3B, c), and there are only red fluorescence dots (from QDs in FMNPs) under excited with ultraviolet light (Figure 3B, d). Interestingly, the red fluorescence dots in Figure 3B(c) keep the same positions with the orange fluorescence dots in Figure 3B(b) while the green fluorescence dots in Figure 3B(c) keep the same positions with the red fluorescence dots in Figure 3B(d), suggesting the bacteria stained with both orange and red fluorescence should be E. coli and the bacteria stained with both green and red fluorescence should be S. typ. These results are consistent with the results in Figure S6. After 25 s and 60 s magnetic separation, S. typ still displayed green and red fluorescent dots and E. coli showed orange and red fluorescent dots, respectively, as shown in Figure 3C. This suggests that apt-E. coli-W-FMNPs and apt-S. typ-S-FMNPs could capture and detect their corresponding targets of E. coli and S. typ from a mixed sample under an external magnetic field, respectively. To further validate that the bacteria captured by apt-FMNPs were the target bacteria from the perspective of molecular biology, PCR was carried out according to previous papers.39-42 Figure 3D showed the mixture of S. typ, E. coli, S. aureus and L. monocytogenes before separation as four corresponding gene fragments, 605 bp fragment of iroB gene for S. typ, 206 bp fragment of stx2 gene for E. coli, 132 bp fragment of femA gene for S. aureus and 370 bp fragment of prs gene for L. 16


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monocytogenes (lane 2), using each bacterium as positive control (lane 3 , 6, 7 and 8). After adding apt-FMNPs, the captured specimens at 25 s and 60 s were successively subjected to PCR analysis, respectively, as done as the pure bacteria. Under these conditions, iroB and stx2 gene still displayed in the agarose gel without femA and prs gene (lane 4 and 6 in Figure 3D), indicating that S. typ and E. coli were successfully and specifically captured. Collectively, these results suggested that the apt-FMNPs nanoprobes could identify and capture S. typ and E. coli with high reliability and specificity.

Figure 3. Fluorescence spectrum (A) and fluorescence microscope images (B) of the dual mixture of E. coli and S. typ after incubated with the mixture of Cy3-apt-E. coli-W-FMNPs and FAM-apt-S. typ-S-FMNPs simultaneously. (C) Fluorescence microscope images of the pathogen@nanoprobes complexes after capture and magnetic separation from a mixed sample under 25 s and 60 s of magnetic adsorption time points. Left pane: S. typ captured and detected by FAM-apt-S. typ-S-FMNPs. Right pane: E.coli captured and detected by Cy3-apt-E. coli-W-FMNPs. (D) Agarose gel electrophoresis results of PCR products from the bacteria captured with apt-FMNPs. Quantification of Multiple Target Bacteria. Conditions, including time, 17



temperature, and nanoprobes concentration, were optimized (Figure S9), then the linear range and detection limit of apt-FMNPs nanoprobes for target bacteria were studied. Specifically, a series of S. typ and E. coli solutions with different concentrations (0, 10, 101.2, 101.4, 101.6, 101.8, 102, 103, 104, 105, 106, 107 and 108 cfu/mL) were incubated with apt-FMNPs nanoprobes, respectively, followed by separation and determination of the fluorescence intensity of E. coli @apt-E. coli-W-FMNPs and S. typ@apt-S. typ-S-FMNPs complex of each specimen. As shown in Figure S10, the enhanced fluorescence intensity ∆F (∆F=F−F0, where F0 and F stand for the fluorescence intensity of sediment when apt-FMNPs nanoprobes are incubated without or with target bacteria) was correlated with the concentration of S. typ in the range of 63-108 cfu/mL with a detection limit of 25 cfu/mL and correlated with E. coli in the range of 40-108 cfu/mL with a detection limit of 16 cfu/mL (∆F = 359.3log10 NS. typ + 14.9, R2 = 0.9922 and ∆F = 867.7log10 NE. coli + 53.24, R2 = 0.9915, where NS. typ and NE. coli stand for the quantity of S. typ and E. coli in cfu/mL). Near-Simultaneous Detection of S. typ and E. coli in Authentic Samples. In order to demonstrate the practical application of the proposed method for the near-simultaneous detection of multiple bacteria in authentic sample, qualified milk, sterile human serum and human urine samples without dilution spiked with S. typ and E. coli were analyzed. First, the limits of detection for the two bacteria in the three samples were measured. The results showed the detection limits of 150 cfu/mL for S. typ and 100 cfu/mL for E. coli in milk, 120 cfu/mL for S. typ and 80 cfu/mL for E. coli in human serum, and 120 cfu/mL for S. typ and 80 cfu/mL for E. coli in human 18


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urine (Figure S11). Further, as shown in Table 1, the apt-FMNPs nanoprobes-based method for detection of S. typ and E. coli showed good recovery, indicating that the proposed magnetism-resolved-based strategy could be applied to near-simultaneous detection of two bacteria coexisting in authentic samples. In our opinion, the high recovery efficiency may owe to the following elements. Firstly, aptamers with high affinity and specificity for their targets can make apt-FMNPs nanoprobes specifically capture target bacteria, which could improve the recovery efficiency. Secondly, the strong fluorescence intensity of apt-FMNPs nanoprobes could improve the detection sensitivity. Thirdly, the variable magnetic response of apt-FMNPs nanoprobes is helpful for the effective separation of each target pathogen at different magnetic adsorption time points, which improved the recovery efficiency. A brief comparison of various methods for the detection of S. typ and E. coli is summarized in Table S4. Compared with other methods reported, the proposed strategy in this study presents high sensitivity, wide analytical range and rapidness (within 1h) for the near-simultaneous detection of S. typ and E. coli. Table 1. Recovery efficiency of S. typ and E. coli spiked in milk, human serum, and human urine samples based on the proposed strategy samples

milk

serum

urine

Recovery (%)

RSD (%, n=3)

(Log10 cfu/mL)

S. typ

E. coli

S. typ

E. coli

2.12

2.19

84.9

87.6

5.91

4.30

4.05

4.15

89.9

92.3

2.83

4.00

7.19

7.33

95.9

97.7

1.48

1.10

added S. typ

added E. coli

found S. typ

found E. coli

(Log10 cfu/mL)

(Log10 cfu/mL)

(Log10 cfu/mL)

2.5

2.5

4.5

4.5

7.5

7.5

2.5

2.5

2.23

2.45

89.3

97.9

5.71

3.80

4.5

4.5

4.14

4.52

92.0

100.4

2.80

2.13

7.5

7.5

7.23

7.48

96.4

99.8

1.13

1.23

2.5

2.5

2.13

2.51

85.3

100.5

2.55

3.97

4.5

4.5

4.21

4.57

93.6

101.6

4.56

1.99

7.5

7.5

7.33

7.48

97.7

99.7

1.35

1.17

19



AST Assay of Target Pathogens Separated from Urine Samples. AST is typically used for the rapid identification and management of effective antimicrobial agents in the treatment of infectious diseases caused by clinical pathogens. According to the Clinical and Laboratory Standards Institute (CLSI) criteria,43 AST assay results usually categorize an antimicrobial agent according to 3 levels, including “susceptible (S)”, “intermediate (I)”, and “resistant (R)”, respectively. It is highly desirable that pathogens in clinical samples could be subjected to AST immediately after identification. Unfortunately, most of the current strategies for AST involve time-consuming and complex separating procedures, particularly facing to samples containing different pathogens.19 Since the multiple bacteria assay strategy proposed in this study could sequentially separate each target bacterium, we envision that AST assay for the bacterial target of interest should be conducted directly. To exclude the impact of apt-FMNPs on AST results, the cytotoxicity of apt-FMNPs to bacteria was investigated by the plate-counting method prior to AST. As shown in Figure S12, bacterial viability remained about 100% during incubation with apt-FMNPs nanoprobes at successive incubation time points of 0, 0.5, 1, 3, 6, 12 and 18 h, indicating that our apt-FMNPs have no obvious effect on the viability of the target bacteria. Then the target pathogens isolated from human urine samples with aptFMNPs nanoprobes were directly subjected to AST with four antibiotic drugs, including Tet, Amp, Gen, and Nal, respectively. To make AST results convincing, pure S. typ and E. coli bacteria were employed as positive control references and subjected to AST under the same conditions. As shown in Figure 4 and Table S5, both 20


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pure S. typ and S. typ@apt-S. typ-S-FMNPs presented resistance to Tet and sensitivity to Amp, Gen and Nal (Figure 4A and B), and both pure E. coli and E. coli@apt-E. coli-W-FMNPs showed similar resistance to Tet, intermediate resistance to Amp and sensitivity to Nal and Gen (Figure 4C and D). The AST assay for pure S. aureus (Figure 4E) and pure L. monocytogenes (Figure 4F) showed no non-target bacteria were captured by the apt-FMNPs nanoprobes. It could be analyzed that if the S. aureus captured by apt-FMNPs nanoprobes, the mean diameter of the inhibition zone for Tet in the sample of S. typ@ apt-S. typ-S-FMNPs and E. coli@ apt-E. coli-W-FMNPs should be greater than 14 mm (according to Table S5), and if the L. monocytogenes captured, the mean diameter of the inhibition zone for Tet, Amp, and Gen in the sample of S. typ@ apt-S. typ-S-FMNPs and E. coli@ apt-E. coli-W-FMNPs also should be greater. These results demonstrated that the multiple target pathogens specifically recognized and rapid sequentially captured by apt-FMNPs nanoprobes-based strategy are ready for the downstream AST assay, which is a formidable step forward compared with the current multiple pathogen detection strategies.44

Figure 4. Photographs of the cultured agar plates of AST assay (A: S. typ, B: S. typ@ apt-S. typ-S-FMNPs, C: E. coli, D: E. coli@ apt-E. coli-W-FMNPs, E: S. aureus, F: L. monocytogenes). 21



CONCLUSION In summary, a new magnetism-resolved strategy based on magnetic-encoded

apt-FMNPs nanoprobes was successfully developed for the near-simultaneous, rapid and specific detection of multiple pathogenic bacteria. The sequential magnetism fished out each target bacterium from authentic sample using the apt-FMNPs nanoprobes and samples were successfully subjected to AST assay. Thus, both multiple pathogen identification and subsequent AST assay will provide essential to the treatment of infectious disease. In general, magnetic-encoded FMNPs based strategy could achieve near-simultaneous detection and separation of multiple pathogenic bacteria interested for downstream studies. Further efforts will be made to improve the properties of FMNPs nanoprobes with a series of gradient and controllable magnetic response, more uniform size and facile preparation for analyzing other multiple targets like heterogeneous phenotypes of circulating tumor cells, virus and so on.

ASSOCIATED CONTENT

Supporting information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Figures S1−S12 and Tables S1−S5 with brief explanations.

AUTHOR INFORMATION

Corresponding Author 22


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*E-mail: [email protected].

Fax: (+86)2368251225

Author Contributions All authors contributed equally to the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS

This work was supported by the National Natural Science Foundation of China (21575118, 21622704), Fundamental Research Funds for the Central Universities (XDJK2017A017, XDJK2018AA007), and funding support from Beijing National Laboratory for Molecular Sciences (BNLMS).

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Magnetism-Resolved Separation and Fluorescence Quantification for

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