A Highly Specific Bacteriophage-Affinity Strategy for Rapid Separation

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A Highly Specific Bacteriophage-Affinity Strategy for Rapid Separation and Sensitive Detection of Viable Pseudomonas aeruginosa Yong He, Mengyao Wang, Enci Fan, Hui Ouyang, Huan Yue, Xiaoxiao Su, Guojian Liao, Lin Wang, Shuguang Lu, and Zhifeng Fu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04389 • Publication Date (Web): 23 Jan 2017 Downloaded from http://pubs.acs.org on January 24, 2017

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Analytical Chemistry

A Highly Specific Bacteriophage-Affinity Strategy for Rapid Separation and Sensitive Detection of Viable Pseudomonas aeruginosa Yong He,†,‡ Mengyao Wang,† Enci Fan,† Hui Ouyang,† Huan Yue,† Xiaoxiao Su,† Guojian Liao,† Lin Wang,† Shuguang Lu,║,* Zhifeng Fu†,* †

Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Ministry of

Education), College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China ‡

Department of Pharmacy, Affiliated Hospital of Zunyi Medical College, Zunyi

563000, China ║

Department of Microbiology, College of Basic Medical Science, Third Military

Medical University, Chongqing 400038, China

* Corresponding author. Tel.: +86-23-6825-2243; Fax: +86-23-6825-2834. * Corresponding author. Tel.: +86-23-6825-0184; Fax: +86-23-6825-1048. E-mail address: [email protected] (S.G. Lu), [email protected] (Z.F. Fu)

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ACS Paragon Plus Environment


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ABSTRACT: A virulent bacteriophage highly specific to Pseudomonas aeruginosa (P. aeruginosa) was isolated from hospital sewage using a lambda bacteriophage isolation protocol. The bacteriophage, named as PAP1, was used to functionalize tosyl-activated magnetic beads to establish a bacteriophage-affinity strategy for separation and detection of viable P. aeruginosa. Recognition of the target bacteria by tail fibers and baseplate of the bacteriophage led to capture of P. aeruginosa onto the magnetic beads. After a replication cycle of about 100 min, the progenies lysed the target bacteria and released the intracellular adenosine triphosphate. Subsequently, firefly luciferase-adenosine triphosphate bioluminescence system was used to quantitate the amount of P. aeruginosa. This bacteriophage-affinity strategy for viable P. aeruginosa detection showed a linear range of 6.0 × 102 - 3.0 × 105 CFU mL-1, with a detection limit of 2.0 × 102 CFU mL-1. The whole process for separation and detection could be completed after bacteria capture, bacteriophage replication and bacteria lysis within 2 h. Since the isolated bacteriophage recognized the target bacteria with very high specificity, the proposed strategy did not show any signal response to all of the tested interfering bacteria. Furthermore, it excluded the interference from inactivated P. aeruginosa because the bacteriophage could replicate only in viable cells. The proposed strategy had been applied for detection of P. aeruginosa in glucose injection, human urine and rat plasma. In the further work, this facile bacteriophage-affinity strategy could be extended for detection of other pathogens by utilizing virulent bacteriophage specific to other targets.

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A substantial toll is being exacted continuously on health and health-care resources by infectious diseases.1 As one of the top three causes of opportunistic infectious bacteria,2 infection with transmissible strains of Pseudomonas aeruginosa (P. aeruginosa) in adult with cystic fibrosis is associated with high risk of mortality.3 P. aeruginosa also accounts for paediatric hospital-acquired bacteraemia besides nosocomial infections in neonatal intensive care units in many undeveloped countries.4-5 Due to the potential pathogenicity of P. aeruginosa, it is essential to develop facile, rapid, specific and sensitive methods for detection of the notorious pathogen. Currently, many methods based on bacterial culture and colony counting,6 polymerase chain reaction7-9 and molecular recognition10-13 have been developed to detect pathogenic bacteria. As a “gold standard”, the conventional bacterial culture and colony counting-based method is accurate, sensitive and reproducible, but still labor-intensive and time-consuming.14 As an alternative approach to the conventional method, polymerase chain reaction-based method shows remarkable advantages in specificity, sensitivity and rapidity. Unfortunately, besides requiring skilled manipulation, it often suffers from false positive resulting from exogenous pollutants. Furthermore, it cannot distinguish viable bacteria from inactivate bacteria.15 Molecular recognition-based methods, such as immunoassay and aptasensor,16-17 possess the advantages of simple manipulation and short assay time, whereas cannot distinguish viable bacteria from inactivate bacteria14 and still demand expensive biomaterial-based molecular recognition agents. Other molecular recognition agents such as antibiotic18 and lectin19 have attracted increasing interest for bacteria detection due to their low cost. Nevertheless their application was obviously limited by the lack of specificity for distinguishing different bacteria. 3



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Bacteriophages are well known to be viruses that specifically infect their target bacteria. The replication cycle of virulent bacteriophages involves multiple steps. Specific recognition of the target bacteria is initialed with reversible binding by bacteriophage tail fibers and followed by baseplate positioning. Then the bacteriophages irreversibly bind to a secondary receptor of the target bacteria through a different tail protein. Thereafter, positioning of the tail on bacterial surface triggers DNA injection into the target cells, followed by progenies assembling utilizing the replicative machinery of target bacteria. With the aid of holin, a bacteriophage protein accumulating in the bacterial inner membrane to form pores allowing the endolysin to reach the peptidoglycan layer, endolysin lyses the bacterial cell resulting in the release of mature progenies and intracellular substances of the target bacteria.20 Bacteriophages show immense potentials to form the foundation to develop specific diagnostic and therapeutic tools for bacterial infection.21-22 For example, bacteriophage amplification technique has been used to detect Staphylococcus aureus (S. aureus) by mass spectrometric quantitation of the released progenies produced by

15

14

N capsid protein of

N-labeled bacteriophages in normal culture media. This

protocol requires complicated isotope labeling of bacteriophage capsid protein and expensive instrumentation.23 As for reporter bacteriophage or bacteriophage display technique, a recombinant bacteriophage needs to be constructed by cloning a gene or DNA sequence into the bacteriophage capsid protein gene, which demands complicated and sophisticated gene manipulation.24-25 Adenosine triphosphate (ATP) exists in all bacteria as the basic energy substance, and its amount in viable cell remains rather constant (~10-18 mol/cell).26 Thus firefly luciferase-ATP bioluminescence (BL) reaction can be used to quantitate bacteria by chemically extracting ATP from the cells.27-28 However, this strategy is lack of 4


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specificity, thus cannot distinguish different bacteria and exclude the interference from complicated sample matrix. In this work, a virulent bacteriophage highly specific to P. aeruginosa was successfully isolated from hospital sewage by a lambda bacteriophage isolation protocol. The bacteriophage named as PAP1 was used as a recognition agent to functionalize tosyl-activated magnetic beads (MBs). With the PAP1-functionalized MBs, a facile bacteriophage-affinity strategy was developed to specifically separate and lyse P. aeruginosa to release intracellular ATP for BL detection of P. aeruginosa. EXPERIMENTAL SECTION Instrumentations. The BL signal detection was performed on a multifunctional chemiluminescence analyzer (Xi’an Remax Electronic Science & Technology Co., Ltd., China). The scanning electron micrographs were obtained from a S-3000N scanning electron microscope (SEM) (Hitachi Instrument Co., Ltd., Japan). The transmission electron micrographs were obtained from a Tecnai 10 transmission electron microscope (TEM) (Royal Dutch Philips Electronics Ltd., The Netherlands). Fluorescence micrographs were obtained from a LSM710 laser confocal fluorescence microscope (Carl Zeiss Co., Ltd., Germany). Ultrapure water (18.2 MΩ) was purified by an ELGA PURELAB classic system (VWS Ltd., UK). Chemicals and Materials. Tosyl-activated MBs with a diameter of 1.5 µm was obtained from JSR Life Sciences (Japan). Recombinant luciferase was purchased from Promega (USA). D-luciferin and fluorescein isothiocyanate (FITC) were provided by Sigma-Aldrich Chemical Co., Ltd. (USA). Bovine serum albumin (BSA) was purchased from Gibco (USA). Ethylenediamine tetraacetic acid disodium salt and 1-4-dithiothreitol were obtained from Kelong Chemical Co., Ltd. (China). Tryptone and

yeast

extract

were

obtained

from

5


Oxoid

Ltd.

(USA).


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2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris) was purchased from Aladdin Reagent Ltd. (China). Polyethylene glycol 8000 was provided by Boer Biotech Co., Ltd. (China). The 96-well polystyrene microplate was purchased from Corning Incorporated (USA). Strains of Salmonella typhimurium (S. typhimurium) (GIM 1.237), Escherichia coli (E.coli) (GIM 1.223), Shigella dyseteriae (S. dyseteriae) (GIM 1.236), Pseudomonas solanacearum (P. solanacearum) (GIM 1.77), Pseudomonas putida (P. putida) (GIM 1.445) and Staphylococcus epidermidis (S. epidermidis) (GIM 1.444) were provided by Guangdong Microbiology Culture Center (China). Strains of Streptococcus mutans (S. mutans) (CCTCC AB 99010) and S. aureus (CCTCC AB 91093) were obtained from China Center for Type Culture Collection. Human urine was obtained from healthy adult volunteers. Rat plasma was obtained from Sprague-Dawley rat (Chongqing Academy of Chinese Materia Media, China). 5% glucose injection (Southwest Pharmaceutical Co., Ltd., China) was purchased from local hospital. The ATP BL solution was 25 mM Tris buffer (pH 7.8) containing 0.075 mg mL-1 luciferase, 0.25 mg mL-1 D-luciferin, 2.5 mM ethylenediamine tetraacetic acid disodium salt, 25 mM Mg(Ac)2, 2.5 mM 1-4-dithiothreitol and 1.0 mg mL-1 BSA. The Luria-Bertani (LB) broth was composed of 10 g L-1 tryptone, 10 g L-1 NaCl and 5.0 g L-1 yeast extract. The semisolid LB broth was composed of 10 g L-1 tryptone, 10 g L-1 NaCl, 5.0 g L-1 yeast extract and 0.67 g L-1 agar. The separation buffer and dilution buffer for P. aeruginosa was 10 mM phosphate buffer saline (PBS) at pH 7.4. The coupling buffer for MBs functionalization was 0.10 M borate buffer at pH 9.5. The blocking buffer was 25 mM Tris buffer (pH 9.5) containing 0.1% BSA. The lysis buffer was 25 mM Tris buffer at pH 7.4. Preparation of P. aeruginosa. According to standard protocol for bacterial culture 6


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and counting, P. aeruginosa was grown in LB broth to logarithmic phage for 6 h at 37 °C under constant shaking at 180 rpm (OD600 = 0.3 - 0.4). Standard LB agar plate counting was used to evaluate the concentration of bacteria. For modeling nutrient-poor (NP) condition of P. aeruginosa, the bacterial culture was centrifuged at 4000 g for 2 min to completely remove the culture media, and the collected pellet was resuspended in dilution buffer overnight at room temperature (RT). Prior to BL detection of P. aeruginosa, all samples were stored at 4 °C for 45 min to adjust the bacteria into a homogeneous state of extremely slowing growth. Then the cooled samples were spiked with 50 µL of pre-cooled LB broth so that even NP bacteria were adjusted to nutrient-rich (NR) condition. Isolation of PAP1. PAP1 was isolated from the sewage of Southwest Hospital (Chongqing, China) based on a lambda bacteriophage isolation protocol,29 as detailedly described in supporting information. The plaque forming units were recognized as successful isolation of bacteriophage PAP1. The bacteriophage titer was measured by standard plaque assay. TEM Characterization of PAP1. Twenty microliters of PAP1 solution at 1.2 × 1012 PFU/mL was deposited onto a carbon-coated copper grid, followed by being stained with the same volume of 2.0% phosphotungstic acid for 1 min at RT. Then the stained PAP1 was observed by TEM to obtain its size and morphology. Preparation of FITC-Tagged PAP1. Two hundred microliters of PAP1 at 1.2 × 1012 PFU mL-1 was mixed with 1.0 mg of FITC, followed by a 12-h reaction at 4 °C. Then the reaction was ended by adding 20 µL of 4.0 M glycine solution. The obtained solution was dialyzed in 10 mM PBS at pH 7.4 for 48 h at RT. Laser Confocal Microscope Imaging of Fluorescence-Stained P. aeruginosa. One 7



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hundred microliters of P. aeruginosa solution at 2.0 × 106 CFU mL-1 was incubated with 500 µL of 1.2 × 1012 PFU mL-1 FITC-tagged PAP1 solution at 37 °C for 20 min. The stained P. aeruginosa cells were washed thrice and resuspended in 1.0 mL of 10 mM PBS. Then 5.0 µL of the obtained solution was dropped onto a microscope slide, and covered by a coverslip. The prepared specimen was observed under the laser confocal fluorescence microscope. The excitation and emission wavelengths for FITC were set to be 488 nm and 525 nm, respectively. Conjugation of PAP1 onto Tosyl-Activated MBs. One hundred microliters of tosyl-activated MBs were washed twice with 1.0 mL of coupling buffer, and then resuspended in 900 µL of the same buffer. After that, 100 µL of PAP1 solution at 2.7 × 1011 PFU mL-1 was added into the MBs suspension, followed by an 8-h incubation at 37 °C and a subsequent 16-h incubation at RT under gentle shake at 20 rpm. The obtained PAP1-functionalized MBs were washed twice with 1.0 mL of dilution buffer and blocked overnight with 1.0 mL of blocking buffer. The blocked MBs were then washed and resuspended in 1.0 mL of dilution buffer. Detection of P. aeruginosa. One milliliter of P. aeruginosa standard sample (for calibration curve) or practical sample and 20 µL of PAP1-functionalized MBs were mixed together and incubated for 25 min at 37 °C under under gentle shake at 60 rpm. The obtained MBs bearing P. aeruginosa was magnetically separated, and treated with 400 µL of lysis buffer to disrupt the cells of P. aeruginosa. Lastly, 50 µL of the obtained lysate was injected into a microplate, and reacted with 50 µL of ATP BL solution to trigger the signal for the quantitation of P. aeruginosa. RESULTS AND DISCUSSION Biological Characterization of Isolated PAP1. According to the lifestyle, bacteriophages can be classed into temperate bacteriophages and virulent 8


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bacteriophages. For temperate bacteriophages, they initiate a lysogenic cycle in which their genome often integrates into the host genome to assume a quiescent state named as prophage. In contrast, virulent bacteriophages can replicate only through a lytic cycle. After the bacteriophages infect the target bacteria, the bacteriophages utilize the host metabolism and molecular machineries for generating the progenies.20,30 As seen in Fig. 1A, the transparent plagues on the lawn of P. aeruginosa showed successful isolation of PAP1. This result also demonstrated that the isolated PAP1 was a virulent bacteriophage that could lyse the target bacteria to generate the progenies under suitable conditions for bacteria growth. The isolated PAP1 was observed using TEM. As shown in Fig. 1B, it was composed of an icosahedral symmetrical head with a size of around 70 nm and a tail with a length of around 130 nm. Using the classical one-step growth curve protocol,20 its capture time and complete lysis time were measured to be around 15 min and 60 min, respectively, after PAP1 and P. aeruginosa were mixed together at RT. Principle of Bacteriophage-Affinity Strategy for P. aeruginosa Detection. As illustrated in Scheme 1, PAP1 was utilized as the specific recognition agent to functionalize MBs by a chemical reaction between amino group of bacteriophage protein and tosyl on MBs. After the cells of P. aeruginosa were specifically captured and separated by PAP1-functionalized MBs, the captured cells were injected with bacteriophage DNA. Then progenies were replicated by utilizing the replicative machinery of the captured cells. After progenies maturation, the target bacteria were lysed to release the progenies and the intracellular ATP. Lastly, firefly luciferase-ATP BL system was adopted to detect the level of ATP in the lysate to quantitate P. aeruginosa. Capture and Lysis of P. aeruginosa by PAP1-Functionalized MBs. In order to 9



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investigate the capture ability of the isolated bacteriophages, PAP1 was tagged with FITC and incubated with P. aeruginosa. As shown in Fig. 2, after P. aeruginosa was stained by FITC-tagged PAP1, green fluorescence from FITC was observed on the cell of P. aeruginosa. This phenomenon demonstrated the binding behavior between PAP1 and P. aeruginosa. SEM and standard plaque assay were used to evaluate the capture and lysis ability of PAP1 functionalized on MBs. As seen in Fig. 3A, PAP1-functionalized MBs resulted in transparent plagues on the lawn of P. aeruginosa, which demonstrated that the chemically conjugated PAP1 still retained the infectious activity. In a following long-term investigation, the infectious activity was found to remain unchanged for 6 months when the prepared PAP1-functionalized MBs were stored in PBS at 4 °C. After PAP1-functionalized MBs were incubated with the target bacteria, they were magnetically separated and observed using SEM. As shown in Fig. 3B, cells of P. aeruginosa were bound to the surface of PAP1-functionalized MBs. Interestingly, holes were clearly observed on some cells of P. aeruginosa (Fig. 3C), which were probably induced by the synergetic action of bacteriophage holin and endolysin.20,31 After the progenies matured, the holin aggregated to form structures in the inner membrane which allowed the endolysin out to degrade the cell wall.20 Thus progenies and intracellular substances including ATP could be released from the formed holes. This phenomenon demonstrated the lysis ability of PAP1-functionalized MBs, which facilitated detection of P. aeruginosa utilizing firefly luciferase-ATP BL system. Optimization of Capture Time and Lysis Time. The capture time and the lysis time can remarkably influence the amounts of captured P. aeruginosa cells and released intracellular ATP. Thus the two parameters were optimized by using P. aeruginosa standard solution at 1.0 × 105 CFU mL-1. As shown in Fig. 4, the maximal capture 10


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time and the complete lysis time of PAP1-functionalized MBs for P. aeruginosa were around 30 min and 100 min, respectively, which were longer than those of the unconjugated bacteriophages. The prolonged capture time and lysis time might be partly attributed to the changed environment for replication cycle and the decreased mobile capability of PAP1 after the bacteriophages were conjugated with MBs with a much bigger size of 1.5 µm. For the capture process, the capture amount began to decrease after 30 min because some captured cells were lysed by PAP1 at that time. Detection Performance. As seen in Fig. 5, the proposed method showed a linear range of 6.0 × 102 - 3.0 × 105 CFU mL-1 for P. aeruginosa detection, with a correlation coefficient of 0.9928. The detection limit was 2.0 × 102 CFU mL-1 at a signal to noise ratio of 3. The regression equation for P. aeruginosa detection was lgI (a.u.) = 0.85 + 0.50lgC (CFU mL-1), where I and C were the BL intensity and the concentration of P. aeruginosa, respectively. The relative standard deviation (RSD) values for P. aeruginosa standard samples at low (6.0 × 102 CFU mL-1), medium (3.0 × 104 CFU mL-1), and high (3.0 × 105 CFU mL-1) concentrations were measured to be 3.9%, 7.5%, 2.5%, respectively. Specificity. Three Gram-positive bacteria (S. mutans, S. aureus and S. epidermidis) and three Gram-negative bacteria (S. typhimurium, E. coli, S. dyseteriae) were detected by the proposed method to investigate their potential interference to P. aeruginosa detection. The concentrations of all the tested bacteria were 1.0 × 105 CFU mL-1 in the specificity investigation. The following equation was used to calculate the degree of interference (DI) of the interfering bacteria: DI =

P−B ×100% I −B

(1)

Here, P, I and B represented the BL signals from P. aeruginosa, the interfering bacteria and the blank sample, respectively. 11



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As shown in Fig. 6, the DI values of the tested interfering bacteria were all below 3.1%. The specificity was also assessed using Mixture 1 composed of all the above interfering bacteria, and the obtained DI value was 2.3%. Meanwhile, Mixture 2 was prepared by mixing the six interfering bacteria with P. aeruginosa. The obtained BL response from Mixture 2 showed a minor difference of 4.0% in comparison with that of P. aeruginosa. The potential interference of two other species of Pseudomonas including P. solanacearum and P. putida was also investigated in detail, and the obtained DI values of 2.9% and 2.4% for the two bacteria demonstrated the species specificity of this protocol. All the above results demonstrated that the proposed protocol possessed ideal specificity for recognizing P. aeruginosa. Furthermore, to investigate its ability for excluding interference from inactive P. aeruginosa, P. aeruginosa was sterilized by ultraviolet exposure and detected by the protocol. As shown in Fig. 6, the DI value of the inactivated P. aeruginosa was 3.1%, which demonstrated that the proposed strategy could distinguish viable pathogens from inactive ones.

Influence of Nutrient Condition. The influence of nutrient condition on BL response of P. aeruginosa was investigated by detecting bacterial samples prepared in different media. Nutrient regimes I and II were constructed by preparing 1.0 × 105 CFU mL-1 bacteria in NP (1000 µL of PBS) and NR (1000 µL of LB) media, respectively. Nutrient intermediate (regime III) was constructed by mixing 950 µL of NP bacteria with 50 µL of LB to reach a final bacterial concentration of 1.0 × 105 CFU mL-1 just prior to detection. As shown in Fig. 7, the BL signal of regime I was only 7.8% of that of regime II, which might be attributed to low intracellular ATP level in NP regime. Also seen in this figure, regime III showed a minor response decrease of 3.1% in comparison with regime II. The result indicated that the addition of small amount of 12


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LB into NP regime could increase intracellular ATP level to normal level close to NR regime in the short duration for incubating P. aeruginosa with PAP1-functionalized MBs.

Practical Sample Detection. Glucose injection (5%), human urine, and rat plasma were used as the sample matrixes for P. aeruginosa to estimate its reliability for field detection of pharmaceutical product and point-of-care test of clinical specimen. The samples were spiked with P. aeruginosa standard solution at various given concentrations to perform recovery tests. The results listed in Table 1 showed acceptable recovery values ranging from 77.4% to 96.9%, with all RSD values below 8.9%, demonstrating its reliability for detecting bacteria in complicated sample matrixes.

CONCLUSIONS In summary, PAP1 highly specific to P. aeruginosa was isolated from hospital sewage. One-step growth curve test indicated that it was a virulent bacteriophage with a complete lysis time of around 60 min. Using PAP1-functionalized MBs, highly efficient separation and lysis of viable P. aeruginosa could be conducted in one step. Then the released intracellular ATP could be utilized for highly sensitive BL detection of this pathogen. This strategy showed many advantages such as rapidity, low cost and facile manipulation besides ideal specificity and high sensitivity. Compared with other specific recognition agents such as antibody and aptamer, the bacteriophage could be easily and rapidly prepared on a large scale with low cost in most microbial laboratories. It could distinguish viable P. aeruginosa from other bacteria and inactive P. aeruginosa. This proof-of-principle work could be easily extended to detect other pathogens by using bacteriophages specific to different bacteria. Our further research will focus on exploring bacteriophage-based biosensor array for multiplexed BL 13



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detection of multiple pathogens. Also, we can try to extend this protocol to detect P. aeruginosa in biofilm as this pathogen often grows in this manner.

Acknowledgement This work was financially supported by the Natural Science Foundation of China (21475107).

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(30) Calendar, R. The Bacteriobacteriophages, 2nd ed.; Oxford University Press: UK, 2006. (31) White, R.; Chiba, S.; Pang, T.; Dewey, J. S.; Savva, C. G.; Holzenburg, A.; Poqliano, K.; Young, R. Proc. Natl. Acad. Sci. USA 2011, 108, 798-803.

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Figure Captions Figure 1 (A) Photograph of plague forming units of PAP1 and (B) TEM micrograph of PAP1.

Figure 2 Fluorescence micrographs of P. aeruginosa stained with FITC-tagged PAP1: (A) bright field image, (B) green fluorescence image.

Figure 3 (A) Photograph of plague forming units of PAP1-functionalized MBs, (B) SEM of P. aeruginosa captured by PAP1-functionalized MBs, (C) SEM of P. aeruginosa showing holes induced by PAP1.

Figure 4 Effects of (A) capture time and (B) lysis time on BL response to P. aeruginosa at 1.0 × 105 CFU mL-1. All other conditions were the chosen optimal conditions (n = 3).

Figure 5 (A) BL responses to P. aeruginosa at (a) 0, (b) 6.0 × 102 CFU mL-1, (c) 3.0 × 103 CFU mL-1, (d) 6.0 × 103 CFU mL-1, (e) 3.0 × 104 CFU mL-1, (f) 6.0 × 104 CFU mL-1 and (g) 3.0 × 105 CFU mL-1. (B) Linear curve for P. aeruginosa detection. All conditions were the chosen optimal conditions (n = 3).

Figure 6 Specificity for P. aeruginosa detection. The concentrations of all bacteria were 1.0 × 105 CFU mL-1 (n = 3).

Figure 7 BL responses to 1.0 × 105 CFU mL-1 P. aeruginosa in NP (regime I), NR (regime II), and nutrient intermediate (regime III) conditions (n = 3). For details please refer to text.

Scheme 1 Schematic illustration of bacteriophage-affinity strategy for P. aeruginosa detection.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Scheme 1

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Table 1 Recovery tests for P.aeruginosa spiked in practical samples (n = 3). Sample

Glucose injection

*Human urine

*Rat plasma

Spiked

Found

RSD

Recovery

(CFU mL-1)

(CFU mL-1)

(%)

(%)

1.1 × 105

1.0 × 105

7.4%

94.3%

1.1 × 104

1.0 × 104

6.9%

91.8%

1.1 × 103

1.0 × 103

8.8%

92.3%

1.1 × 105

0.9 × 105

3.5%

84.6%

1.1 × 104

1.1 × 104

8.6%

96.9%

1.1 × 103

1.0 × 103

6.7%

87.1%

1.1 × 105

0.9 × 105

5.9%

86.6%

1.1 × 104

1.0 × 104

2.0%

91.4%

1.1 × 103

0.8 × 103

1.5%

77.4%

* The samples were 10-time diluted.

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TOC only

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A Highly Specific Bacteriophage-Affinity Strategy for Rapid Separation

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