Alkaline phosphatase-responsive fluorescent polymer probe coated surface for colorimetric bacteria detection

Accepted Manuscript Alkaline Phosphatase-responsive Fluorescent Polymer Probe Coated Surface for Colorimetric Bacteria Detection Eun Bi Kang, Zihnil Adha Islamy Mazrad, Akhmad Irhas Robby, Insik In, Sung Young Park PII: DOI: Reference:

S0014-3057(18)30455-5 https://doi.org/10.1016/j.eurpolymj.2018.05.035 EPJ 8433

To appear in:

European Polymer Journal

Received Date: Revised Date: Accepted Date:

7 March 2018 16 May 2018 25 May 2018

Please cite this article as: Kang, E.B., Mazrad, Z.A.I., Robby, A.I., In, I., Park, S.Y., Alkaline Phosphataseresponsive Fluorescent Polymer Probe Coated Surface for Colorimetric Bacteria Detection, European Polymer Journal (2018), doi: https://doi.org/10.1016/j.eurpolymj.2018.05.035

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Alkaline Phosphatase-responsive Fluorescent Polymer Probe Coated Surface for Colorimetric Bacteria Detection

Eun Bi Kanga, Zihnil Adha Islamy Mazradb, Akhmad Irhas Robbyb, Insik Inb,c, Sung Young Park a,b* a

Department of Chemical and Biological Engineering, Korea National University of Transportation, Chungju 380-702, Republic of Korea

b

Department of IT Convergence, Korea National University of Transportation, Chungju 380702, Republic of Korea

c

Department of Polymer Science and Engineering, Korea National University of Transportation, Chungju 380-702, Republic of Korea

*Corresponding author: E-mail: [email protected] (Sung Young Park)

ABSTRACT 1

The present study aimed to develop an enzymatic colorimetric method using a surfaceadsorbing biosensor to detect and kill bacteria in a single, simple, and rapid assay. The phosphorylated fluorescent probe 2-hydroxychalcone (HCAP) conjugated with an adhesive cationic polymer was designed (HCAP-PVP), which yielded greenish-yellow emission in aqueous buffer. Upon introduction of Escherichia coli and Staphylococcus aureus, the phosphate group inside the HCAP was cleaved by endogenous alkaline phosphatase (ALP) and the greenish-yellow emission ratiometrically changed to deep-red emission. This biosensor system detected bacteria over a wide range of bacterial densities (10 1–107 colonyforming units/mL) after 60 min, with similar bacterial detection abilities between aqueousand solid-phase assays. Furthermore, the presence of a quaternary ammonium of dodecane in this system displayed efficient antibacterial activity because of the change in cellular hydrophobic interactions, which enabled this material to act as a dual sensor and killing material. Thus, this system is a novel, rapid, and simple enzymatic sensor with high sensitivity that can be used as a solid-based platform to detect and directly eliminate bacteria.

Keywords: 2-hydroxychalcone, alkaline phosphatase, enzymatic sensor, bacteria detection, antibacterial activity

1. INTRODUCTION 2

Every year, approximately a million people worldwide experience health issues related to bacterial infections; hence, the Food and Drug Administration has focused on studies regarding bacterial sensors and bactericidal agents [1]. From the past few decades, conventional techniques including plating and culturing, polymerase chain reaction (PCR), surface plasmon resonance (SPR), and enzyme-linked immunosorbent assay (ELISA) have been extensively explored to sense pathogenic bacteria; however, these methods require hours to days to obtain reliable results, sophisticated instruments and settings, also further treatment, which are important limitations [2-7]. Comprehensive studies have focused on colorimetric, electroluminescent, and immunomagnetic detection and electrochemical techniques to detect bacteria; however, most of these studies employed organic ligands or antibodies to enable binding to biological analytes, particularly in bacterial cells [8-10]. The two essential requirements for developing effective sensors for pathogen detection are the limit of detection for both environmental or clinical assessment and highly reproducible sensing along with ease of operation, rapidity, sensitivity, portability, and simple preparation. A biosensor with an advanced design that more easily interacts with bacteria without a specific targeting ligand is required to detect pathogens with high sensitivity in both aqueous- and solid-phase assays [11-12]. A free-targeting ligand biosensor can be obtained by considering the chemical structure of bacteria cell outer membranes which contain abundant functional groups, such as phosphate groups or other phospholipids, which are easily deprotonated to produce negative charges [13-15]. Recent advancements in bacterial sensor development have enabled rapid bacteria detection using fluorescent on/off systems along with excellent electrostatic complexation of protonated-amine polymers and anionic phosphate-bacterial surfaces, without the need for extensive biochemical preparation [12,15]. Moreover, bacteria express large amounts of alkaline phosphatase (ALP) which is dominantly located in the periplasmic area of the bacteria surface wall and is associated with the outer membrane. ALP is 3

commonly used in the clinical setting, particularly for biological assays, and to determine disease severity, but shows limitations in bacteria detection [16]. In 1998, a monitoring system for pathogens through luminescence-based bacterial ALP analysis was developed by Charm Science, Inc. (Lawrence, MA, USA); however, this method has high energy requirements, reducing the assay sensitivity [17]. Furthermore, there are no colorimetric methods for detecting existing contaminating microorganisms based on ALP activity; therefore, such a design is necessary to develop a suitable enzymatic-sensitive biosensor and provide opportunities for establishing a new generation of biosensor technology. Designing different types of fluorescence-based sensors with two-well emission wavelengths can provide a dynamic range of fluorescence measurements. Recently, studies of aggregation-induced emission (AIE) fluorogens as tunable fluorescent agents in biosensing and bioimaging have received wide attention, particularly for detecting DNA, proteins, and sugars with high sensitivity [18-21]. Furthermore, excited-stated intramolecular proton transfer (ESIPT) processes are sensitive to the photochemistry of intramolecular hydrogen bond formation [22]. Combining the two phenomena with modified fluorophores enables ratiometric measurement of fluorescence changes. Some ratiometric probes have been extensively reported, including phosphate-functionalized 2-hydroxychalcone (HCAP) for selective detection of ALP in living cells [23]. In this study, a potential strategy using phosphate-functionalized

2-hydroxychalcone

(HCAP)

conjugated

with

cationic

poly(vinylpyrrolidone) (PVP) was designed as an enzymatic-sensitive bacteria sensor. Moreover, in a solid-phase detection assay, the catechol moieties were functionalized in the probe system (HCAP-PVP) to increase adhesiveness to solid surfaces such as polypropylene (PP). The prepared biosensor showed excellent solubility in solution and produced greenishyellow emission in both the aqueous and solid states. Upon introduction of Escherichia coli and Staphylococcus aureus, the ALP-bacteria disrupted the phosphate groups of HCAP inside the HCAP-PVP and activated the ESIPT and AIE processes simultaneously, inducing changes 4

in the bio-probe from yellow to red emission. Furthermore, in the presence of a quaternary ammonium of dodecane, efficient antibacterial activity was observed because of the contribution of increased hydrophobicity which improved bactericidal activity, enabling this sensor to detect and eliminate bacteria. This colorimetric bacteria-sensing method is a novel, rapid, simple, and rather inexpensive strategy for bacteria detection in both aqueous and coated surface state assays. Moreover, this method does not require further pre-treatment with specific chemicals compared to PCR and ELISA. It also shows excellent antibacterial activity which can be used to directly kill bacteria. Therefore, this system offers a promising strategy for synergistically detecting and killing bacteria for various applications such as biomedical and environmental applications.

2. EXPERIMENTAL SECTION

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2.1 Materials Poly(vinylpyrrolidone) (PVP, MW: 55,000), 2-chloro-3′,4′-dihydroxyacetophenone (CCDP),

2-bromoethylamine,

1-bromododecane,

N-hydroxysuccinimide

(NHS),

ethylcarbodiimide hydrochloride (EDC), ethanol, deionized water, phosphate-buffer saline (PBS), and diethyl ether were utilized as solvents in the chemical reaction. For bacteria experiments, Man–Rogosa–Sharpe (MRS), agar, NaCl, and lysogeny broth (LB) were prepared. Methanol-d4 and deuterium oxide (D2O) were used as NMR solvents. Polypropylene (PP) was chosen as a substrate sample. All materials were obtained from Sigma-Aldrich, South Korea. Phosphate-functionalized 2-hydroxychalcone (HCAP) was prepared as previously reported protocol [23]. 2.2 Characterizations Proton nuclear magnetic resonance (1H-NMR) spectra were observed using a Bruker AVANCE III spectrometer (Waltham, MA, USA) by dissolving the sample in D2O/methanold4 solution. Optical properties were analyzed using an Optizen 2120UV spectrophotometer (Mecasys, Gyeonngi-do, Korea) to measure the absorbance spectra (1 mg/mL sample in DDW) and L550B luminescence spectrometer (Perkin Elmer, Waltham, MA, USA) to obtain the photoluminescence profile. Field-emission scanning electron microscopy (FE-SEM) micrographs were acquired using a JEOL JSM-6700F (Tokyo, Japan). Bacteria were detected using an L550B luminescence spectrometer and Zeiss LSM 510 confocal laser-scanning microscope (Wetzlar, Germany) with 405-, 488-, and 543-nm emission filters and controlled magnification. Particle size was measured by dynamic light scattering on a Zetasizer Nano (Malvern Instruments, Malvern, UK). Static water contact angles were measured with a DO3210 instrument (Krüss, Hamburg, Germany). Flow cytometric analysis (FACS) was conducted using an Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific, Waltham, MA, USA). 6

2.3 Preparation of HCAP-conjugated C/C12-PVP (HCAP-PVP) The

adhesive

cationic

polymer,

CCDP-bromododecane

quaternized

poly(vinylpyrrolidone) [C/C12-PVP], was obtained using a quarternization process [24]. To conjugate HCAP with C/C12-PVP, 2-bromoethylamine was conjugated onto C/C12-PVP using quaternization reaction to obtain an amino group for promoting HCAP binding, and then HCAP was bound through an amine coupling mechanism (EDC/NHS) [25]. In detail, amine-functionalized C/C12-PVP (8.27 × 10-6 mol) was dissolved in 100 mL of DDW, and then activated using EDC (2.85 × 10 -3 mol) and NHS (2.85×10-3 mol) for 15 min. This solution was reacted with HCAP (4.95×10 -4 mol) for 24 h at 22–25°C. The resulting solution was purified by dialysis (MW: 3500) against water and then freeze-dried. 2.4 Preparation of coated PP with HCAP-PVP First, PP film (5 × 5 cm) was cut into small pieces and washed with water and ethanol. Next, a dip-coating technique was performed [12]. Briefly, HCAP-PVP (10 mg/mL) was dissolved in PBS (pH 8.5) and the small pieces of PP film were soaked in HCAP-PVP solution overnight. The PP films were then dried. 2.5 Dual-phase detection assay using HCAP-PVP with bacteria S. aureus and E. coli were cultured in 50 mL MRS and LB media for 24 h at 37C, respectively. After 24 h, dilution was conducted to obtain bacteria solutions of different concentrations (101–107 CFU/mL), and then each bacteria solution was centrifuged to obtain the bacteria pellet. To ensure that the desired concentration value was achieved, the OD 600 was measured with a UV-visible spectrometer and FACS was performed (Table S1). For aqueous-state bacteria detection, 5 mg/mL of HCAP-PVP solution was added to each different bacterial pellet and vortexed for 1 min. The bacteria-labeled HCAP-PVP 7

solutions were incubated in a shaking incubator at 37C for 1 h. After incubation, the bacteria-labeled HCAP-PVP solutions were centrifuged within 5 min, and then washed with PBS three times to remove unreacted HCAP-PVP. The obtained bacteria-labeled HCAP-PVP pellet was dissolved in 1 mL PBS (pH 7.4) and then analyzed with a PL spectrometer. HCAPPVP solution alone was used as a control. In the solid-state assay, bacteria solutions (101–107CFU/mL) were prepared in 2-mL microtubes. The HCAP-PVP-coated PP films were soaked in each solution and incubated in a shaking incubator at 37°C for 1 h. After incubation, the films were removed from each solution and rinsed with PBS (pH 7.4) three times to remove unreacted HCAP-PVP, followed by drying at room temperature (22–25C). Bacteria detection analysis was conducted using a confocal laser scanning microscope.

2.6 Antibacterial activity of HCAP-PVP Antibacterial performance was observed by determining the colony counting-forming units (CFU) for two bacterial strains (S. aureus and E. coli). Briefly, different concentrations of HCAP-PVP were used to treat fixed bacteria solution (107 CFU/mL), and then placed in a shaking incubator at 37°C for 1 h. After incubation, 20 µL of bacteria-labeled HCAP-PVP were dispersed into agar media. The treated agar media for both bacteria were re-incubated for overnight at 37°C and culture growth was calculated. Bacteria solution alone was used as a control to compare growth. 2.7 Scanning electron microscopy (SEM) After the bacteria (S. aureus and E. coli) detection assay, the bacterial suspensions were centrifuged at 4000 rpm (5 min). The bacterial pellets were washed with ethanol (25%, 50%, 75%, and 99%) consecutively [31] and then 100 μL bacteria after treatment was placed

8

on silicon wafers in an oven at 50C for drying. Finally, the silicon wafers containing dried bacteria were observed by FE-SEM.

9

3. RESULTS AND DISCUSSION Because of the high ALP activity in bacteria, a rapid and effective fluorescent probe with antibacterial activity was designed to synergistically detect and kill pathogenic bacteria. HCAP, a fluorescent probe, was combined with an adhesive cationic PVP polymer backbone (HCAP-PVP) containing a quaternary ammonium of dodecane for antibacterial activity. The prepared material was highly soluble in water, yielded greenish-yellow emission, and showed strong adhesive properties on PP film. Scheme 1 illustrates the technique used for bacteria detection in solid-phase assays, utilizing the interaction between HCAP and ALP in bacteria. This sensing mechanism occurs in the presence of endogenic ALP bacteria which can cleave the phosphate group on HCAP sites and convert them to the 2-hydroxychalcone (HCA) form. As a result, the emission color gradually changed from greenish yellow into red emission. Additionally, the combined fluorescent probe showed bacterial killing effects towards S. aureus and E. coli, suggesting simultaneous sensing and antibacterial activity with excellent results. Therefore, this new technique is a novel biosensor for simultaneous sensing of bacteria in a multi-phase assay and effectively kills bacteria in a simple and rapid manner. The chemical compounds of integrated HCAP, C-12 (dodecane), and CCDP in the PVP backbone (HCAP-PVP) were characterized by 1H-NMR (Figure 1). The intense peaks at ~1.6 ppm [3H, -CH3], ~3.4 ppm [1H, −CH], ~1.8-2.2 ppm [2H, −C−CH2], and ~3.4 ppm [2H, N−CH2] indicate the PVP polymer background [12]. The integrated HCAP and CCDP was confirmed at ~6.8–7.6 ppm, indicating the aromatic group of catechol and HCAP molecules [23,26]. Integral peaks of C-12 species at approximately 1−1.3 ppm corresponding to 3H, CH3 and 2H, -CH2 were found, reflecting the successful chemical construction and structural formula of the prepared fluorescent system [27]. The absorption properties of HCAP-PVP were also investigated by UV-Vis spectrometry. As shown in Figure 1(b), intense peaks at 250 and 380 nm were clearly observed, indicating π–π* electron transitions in phenolic 10

compounds of a catechol group [12,26]. The introduction of HCAP onto the polymer resulted in the appearance of a new peak at approximately 416 nm associated with the π–conjugated system in the benzoyl group on HCAP, for which no obvious change was found in the ALP solution. However, the PL of HCAP-PVP showed a distinct shift before and after ALP treatment, as breakage of the phosphate group induced AIE and ESIPT [23,28,29]. In subsequent optical analysis, the PL properties of HCAP-PVP in aqueous solution were determined, as shown in Figure S1. This system showed a maximum peak in the PL spectrum at ~570 nm or a greenish-yellow fluorescent area upon excitation at 510 nm. As shown in Figure 1(c), the diameter of HCAP-PVP particles decreased from an average of 241.2 nm to 189.6 nm, indicating chemical cleavage of the phosphate group by enzymatic ALP activity. Moreover, the TEM images of HCAP-PVP shown in the inset of Figure 1(c) revealed a round-shape morphology, which agreed with the dynamic light scattering data. This may be partly because the long chain of the alkyl forms hydrophobic interactions, which can retain the long chain in the core while the remaining long chain compartment is attached to the shell and shows potential antibacterial activity. The conversion of HCAP to HCA depends on the ALP concentration, which is indicated by a change in the emission color during this period. Therefore, the ALP response of HCAP-PVP should be analyzed at different concentrations of ALP from 0 to 500 U/L using the PL spectra at an excitation wavelength of 510 nm as shown in Figure 1(d). During ALP addition, HCAP catalytically changed to HCA because of phosphate group cleavages. The PL spectra shifted from 570 to 640 nm as ALP concentration increased, indicating simultaneous AIE and ESIPT phenomena. The enzymatic activity reached a maximum for HCAP-PVP at an ALP concentration of 500 U/L with a limit of detection (LOD) at 7.3 U/L, confirming that all phosphate groups had been cleaved [30]. The HCAP-PVP system can be used as a biosensor of ALP activity over a wide range of concentrations. Generally, HCA molecules 11

exhibit strong intramolecular hydrogen bonding to facilitate the ESIPT process, while modifications to the phosphate group cause intramolecular hydrogen bonding that weakens and inhibits the ESIPT mechanism, generating greenish yellow emission [23,30-32]. In ALP solution, the phosphate group on HCAP moieties are released and intramolecular-hydrogen bonding activates the ESIPT and AIE simultaneously. As a result, the ESIPT processes changes the enol form to the keto form in phenolic moieties of HCA and results in the emission of red fluorescence Various techniques have been utilized to detect bacteria including fluorescence on-off systems; here, a new colorimetric strategy was developed with AIE and ESIPT characteristics to detect bacterial contamination based on ALP enzymatic activity. To examine whether this system responds to endogenic bacterial ALP activity, the HCAP system was applied at different concentrations in the bacteria solution (~107 CFU/mL) and the PL spectra obtained at a specific excitation wavelength (510 nm) were analyzed. Because of the different emission properties of HCAP and HCA, in the PL spectra (Figure 2), the emission at 570 nm decreased while that at 640 nm emission wavelengths increased in the presence of high concentrations of bacteria [12,15]. Furthermore, endogenic bacterial ALP which reacted with this system was also observed as shown in Figure 2(c and d). Under UV lamp illumination at 365 nm, the HCAP-PVP showed greenish-yellow emission in the solution, while bacteria treated HCAPPVP strongly emitted a unique red color following degradation depending on the polymer concentration, enabling colorimetric detection of bacteria. Notably, the HCAP-PVP emission was completely quenched because HCAP was hydrolyzed into the HCA because of the ALP enzymatic reaction inside the bacteria. In general, enzymatic-mediated hydrolysis of the HCAP product appeared as a new emission peak at 640 nm; however, bacteria binding inhibits detection of the emission peak as shown in Figure 1(d).

12

Detection performance was optimized using various incubation times with fixed bacteria and nanoparticle concentrations at two specific emission wavelengths. After incubating the bacteria for 1 min, the colorimetric parameters began to change, as shown in the corresponding peak (F/F0) profile (Figure 3(a)). After a short incubation time, our system showed excellent sensing performance towards both strains of bacteria, which was stable for 60 min of incubation. In detail, the HCAP-PVP emission (570 nm) gradually decreased, while the emission of the hydrolyzed form at 640 nm gradually increased depending on the incubation time for both strains of bacteria. Different bacteria concentrations (10 1, 103, 105, and 107 CFU/mL) were treated with fixed concentrations of nanoparticles, as shown in Figure 3(b). At a high concentration of bacteria, ALP activity was increased and more phosphate groups were cleaved in our polymer system, resulting in the disappearance of greenish-yellow emission (570 nm) and increase in red emission (640 nm) depending on the bacteria concentration. To verify the electrostatic binding of HCAP-PVP and surface of bacteria, SEM images of treated bacteria after 60 min of incubation were acquired. Figure 4 shows the noticeable adherence of both bacterial species on the bacteria surfaces, suggesting that the interaction of bacteria and HCAP was influenced by simultaneous hydrophobic and poly-ion electrostatic interactions which were not observed for control bacteria [13,33]. Surface-accessible catechol in this HCAP system offer unique characteristics for use as a film-bacterial sensor based on a colorimetric interaction that can be applied to develop biosensor devices. The HCAP-PVP-coated PP film shown in Figure S2 showed a decrease in the water contact angle, confirming that nanoparticles successfully coated the substrates by reducing the hydrophobicity of the PP film. Similar to in a previous study, a simple and rapid biosensor was developed, specifically for detecting pathogenic bacteria based in a solid-state colorimetric reaction. Monitoring of the behavior of HCAP-PVP on the coated PP film for both bacterial strains at different concentrations was conducted by confocal laser scanning 13

microscopy as shown in Figure 5. As a control, the emission of HCAP-PVP spread across the film surface, while the PP film interacted with the bacteria solution on the film surfaces, and the greenish-yellow fluorescence in the PP film turn to red emission, indicating a strong interaction between the bacteria and cationic site polymer in the film [12]. As shown in Figure 5, the number of bacteria attached to the PP film was increased as bacteria concentration increased, as revealed by the increased red emission intensity, indicating that the enzymatic reaction can be used for a wide range of bacteria concentrations. Over this wide range (101–107 CFU/mL), endogenic ALP levels can control the number of bacteria detected. At high ALP levels, a larger amount of HCAP-PVP can interact with the bacteria, enhancing the red emission of bacteria treated with HCAP-PVP. Based on these results, the colorimetric detection of bacteria was specific for ALP activity in the bacteria. This system showed a remarkable change in emission color when the bacterial ALP concentration increased by cleavage of the phosphate group on HCAP. Furthermore, this system showed a similar ability to detect bacteria both in aqueous solution and on the coated PP surface, making it easy to use for bacteria sensing in aqueous and solid-state assays. Biosensors, particularly those used to sense bacteria, require real-scenario samples to detect contamination. Therefore, contaminated hand was used as actual contaminated samples to test our prepared biosensor and DDW as a control sample (Figure 6). Detection performance over time as determined by confocal microscopy revealed that the results of detection bacteria in contaminated hand samples showed remarkable red emission, as shown in Figure 6. The appearance of red emission on the solid biosensor indicated endogenic ALP activity of bacteria towards the HCAP compartment. It was found that the bacteria performance was time-dependent, as confirmed by the increased red emission with bacteria attachment. The results revealed a different profile for detection in DDW water which was uncontaminated, with no bacteria colonies surviving in the media. Therefore, our nanoparticles 14

are suitable for detecting bacteria from various environments with high accuracy and specificity and showed excellent agreement between solution and real-life samples. This cationic polymer system consists of a long alkyl dodecane, which has been shown to be very efficient for killing bacteria. To evaluate the ability of this system for bacteria removal, the antimicrobial activities were examined using the colony counting technique as shown in Figure 7. When bacteria (107 CFU/mL) were treated at various HCAP-PVP concentrations (0.1–10 mg/mL), a high concentration of polymer (5 and 10 mg/mL) was associated with lower bacterial cell viability (<10%). Based on the values obtained from these investigations, this concentration number is sufficient to inhibit pathogenic bacteria growth. Other bacteria concentrations (105 and 106 CFU/mL) were also evaluated as shown in Figure S3. Each bacteria concentration was treated with 5 mg/mL HCAP-PVP solution, with bacteria solution alone used as a control. The result showed that HCAP-PVP can kill bacteria at different concentrations, supporting the antibacterial activity of this material because of the presence of the quaternary ammonium of dodecane. The antibacterial mechanism of quaternary ammonium of dodecane depends on the change in hydrophobicity of the bacteria cell. Most bacteria outer membranes are negatively charged because of the presence of anionic groups (such as phosphate and carboxyl) in the cell wall. Quaternary ammonium of dodecane interacted with the negative charge of the bacterial surface via ionic interactions. Moreover, the dodecane alkyl chain generated hydrophobic interactions that triggered membrane disruption. Because of this interaction, bacteria cells lost their integrity, resulting in the loss of essential intracellular components such as K+, causing cellular damage to the bacteria [34, 35]. Based on our data, our synergetic biosensor and antibacterial material can be used to overcome the limitations of conventional detection methods. This system can be used to reduce environmental contamination through its antibacterial activity and rapid colorimetric detection of bacteria, although only a small part on environmental contamination. 15

However, this system provides a promising new approach for simply and rapidly detecting and killing bacteria in aqueous and solid assays. Moreover, this system can be further developed as a coatable and recyclable material for various applications such as water treatment and purification processes.

16

4. CONCLUSIONS In conclusion, an HCAP-conjugated adhesive cationic polymer (HCAP-PVP) was prepared using a quaternization and amine-coupling method. The HCAP-PVP showed greenish-yellow emission, indicating the response to ALP activity following cleavage of the phosphate group in HCAP-PVP. Remarkably, in the presence of ALP (0–500 U/L), the emission color of this biosensor changed from greenish-yellow to red, which also occurred when HCAP-PVP was treated with various bacteria concentrations (101–107 CFU/mL). Furthermore, we took advantage of the catechol moieties in the polymer system to design a solid-phase bacteria sensor by coating HCAP-PVP onto the PP film surface. This coated surface assay also showed a change on colorimetric behavior in the presence of bacteria, indicating a similar ability as the aqueous-state assay for detecting bacteria. This fluorescent probe can be applied for killing bacteria with very efficient antibacterial activity because of the contribution of the quaternary ammonium of dodecane. Thus, this ALP-sensitive sensor shows high potency for colorimetry-based bacteria sensing with high sensitivity and versatility in aqueous and solidbased platforms, followed by direct killing of bacteria which is relatively simple, rapid, and inexpensive.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (S.Y.P.) Declaration of Interest The authors declare that they have no competing financial interests.

17

ACKNOWLEDGEMENTS This research was supported by Grant No. 10062079 and R0005237 from the Ministry of Trade, Industry & Energy (MOTIE) and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2017R1A2B2002365).

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22

Scheme 1. (a) Methods of HCAP-PVP synthesis. (b) Illustration of HCAP-PVP application in bacteria detection analysis.

23

Figure 1. (a) 1H-NMR spectra of HCAP-PVP, (b) UV-vis spectra of HCAP-PVP (1 mg/mL). (c) Dynamic light scattering analysis of HCAP-PVP (1 mg/mL) without and with ALP solution, Inset Figure: TEM images of HCAP-PVP. (d) PL spectra of HCAP-PVP (5 mg/mL) under buffer solution and different concentration of ALP (0–500 U/L) at 510 nm of excitation wavelength.

24

Figure 2. Photoluminescence spectra of (a) E. coli treated with HCAP-PVP and (b) S. aureus labeled with HCAP-PVP in various concentrations. Control is 5 mg/mL HCAP-PVP alone and the bacterial concentration was 107 CFU/mL. Photograph of aqueous-state bacteria detection using HCAP-PVP in the presence of (c) E. coli and (d) S. aureus under 365-nm UV lamps. “C” indicates HCAP-PVP as a control at concentrations of 0.1–10 mg/mL and “B” indicates bacteria-labeled HCAP-PVP (0.1–10 mg/mL).

25

Figure 3. PL spectra of bacteria (S. aureus and E. coli) treated HCAP-PVP at two specific emission wavelengths (570 and 640 nm) at an excitation wavelength of 510 nm (a) for different incubation times and (b) different concentrations of bacteria (10 1, 103, 105, and 107 CFU/mL) for 60 min.

26

Figure 4. SEM images of HCAP-PVP (5 mg /mL) incubated with bacteria at 107 CFU/mL for 60 min.

27

Figure 5. Confocal images of PP coated with HCAP-PVP (5 mg/mL) after interaction with bacteria (a) S. aureus and (b) E. coli with after incubation with different bacterial concentrations (101, 103, 105, and 107 CFU/mL) for 60 min.

28

Figure 6. Effectiveness of HCAP-PVP for bacterial detection in solid-phase assay under real sample testing by confocal images. Top: control DDW, Bottom: Contaminated hands.

29

Figure 7. Colony-forming units of E. coli and S. aureus obtained after treatment with various concentrations of HCAP-PVP (0.1, 0.5, 1, 5, 10 mg/mL).

30

Supporting Information

Alkaline Phosphatase-responsive Fluorescent Polymer Probe Coated Surface for Colorimetric Bacteria Detection

Eun Bi Kanga, Zihnil Adha Islamy Mazradb, Akhmad Irhas Robbyb, Insik Inb,c, Sung Young Park a,b* a

Department of Chemical and Biological Engineering, Korea National University of Transportation, Chungju 380-702, Republic of Korea

b

Department of IT Convergence, Korea National University of Transportation, Chungju 380702, Republic of Korea

c

Department of Polymer Science and Engineering, Korea National University of Transportation, Chungju 380-702, Republic of Korea

*Corresponding author: E-mail: [email protected] (Sung Young Park)

31

Figure S1. PL spectra of HCAP-PVP in Tris buffer solution.

32

Figure S2. Contact angle of PP bare, PP coated with C-PVP, C/C12-PVP, HCAP- PVP, and HCAP-PVP with adding ALP (500 U/L). Data are presented as the average ± standard deviation (n = 3).

33

Figure S3. Colony-forming unit of E. coli and S. aureus (105 and 106 CFU/mL) after treatment with 10 mg/mL of HCAP-PVP.

34

Table S1. OD600 measurement and flow cytometry analysis (FACS) for determining bacteria solution concentration. Bacteria E. coli

S. aureus

Concentration (CFU/mL) 107 105 102

OD600 (CFU/mL) 1.36  107 -

FACS (CFU/mL) 1.3015  107 1.1202  105 1.1257  102

107 105 102

1.19  107 -

1.2279  107 1.3567  105 1.2954  102

35

HIGHLIGHTS:  A colorimetric method was designed for detection of bacteria based on ALP activity. 

HCAP-conjugated adhesive cationic polymer showed fluorescent changes in response to ALP.



Quaternary ammonium of dodecane in this system provided antibacterial activity.



This system can be used for both aqueous- and solid-phase assays.



This sensor showed excellent bacteria detection ability and antibacterial activity.

36

Graphical Abstract

37