TCF-ALP: a fluorescent probe for the selective detection of Staphylococcus bacteria and application in “smart” wound dressings

Alkaline phosphatase (ALP) is an important enzyme-based biomarker present in several bacterial species; however, it is currently undervalued as a strategy to detect pathogenic bacteria. Here, we explore our ALP-responsive colorimetric and fluorescent probe (TCF-ALP) for such applications. TCF-ALP displayed a colorimetric and fluorescence response towards Staphylococcus aureus (S. aureus), with a limit of detection of 3.7 × 106 CFU mL−1 after 24 h incubation. To our surprise, TCF-ALP proved selective towards Staphylococcus bacteria when compared with Enterococcus faecalis (E. faecalis), and Gram-negative P. aeruginosa and E. coli. Selectivity was also seen in clinically relevant S. aureus biofilms. Owing to the high prevalence and surface location of S. aureus in chronic wounds, TCF-ALP was subsequently encapsulated in polyvinyl alcohol (PVA)-based hydrogels as a proof-of-concept “smart” wound dressing. TCF-ALP hydrogels were capable of detecting S. aureus in planktonic and biofilm assays, and displayed a clear colour change from yellow to purple after 24 h incubation using ex vivo porcine skin models. Overall, TCF-ALP is a simple tool that requires no prior knowledge, training, or specialist equipment, and has the potential to overcome issues related to invasive swabbing and tissue biopsy methods. Thus, TCF-ALP could be used as a tool to monitor the early development of infection in a wound and allow for the rapid provision of appropriate treatment for Staphylococcal bacterial infections.


Synthesis of TCF-ALP
TCF-ALP was synthesised as outlined in Gwynne et al. [1] Stock solutions of TCF-ALP were stored in DMSO at 4 °C until required.

Preparation of TCF-ALP based PVA hydrogels
A 10% w/v PVA solution was prepared by dissolving poly vinyl alcohol (PVA) in 50 mM Tris HCl (pH 9.2). After the resultant solution had cooled to room temperature, aliquots of 1 mL were transferred into a 12-well microtiter plate and 38.5 µL of TCF-ALP (2.6 mM in DMSO) added to produce homogenous yellow solutions. These solutions were then placed at -80 °C and underwent one freezethaw cycle to produce mechanically stable hydrogels, which were protected from light and stored at 4 °C prior to use.

Bacterial Strains
S. aureus NCTC 10788 and E. coli NCTC 10418 were acquired from Public Health England (PHE; Porton Down, U.K). All further strains used in this assay were kindly provided by Professor Toby Jenkins, Dr Maisem Laabei and Dr Susanne Gebhard, University of Bath, U.K. A full list of bacterial strains used can be found in Table S1. Methicillin sensitive.
Obtained by culture collection from the University of Brighton (Brighton, UK) [20] E. coli DH5α Laboratory isolate developed by D. Hanahan for cloning use. [21] Enterococcus faecalis (E.

Bacterial Enumeration
Estimation of the total viable count of bacterial cultures were determined using the drop count method as outlined by Miles and Misra [23]. The initial bacterial suspension underwent a series of 10-fold dilutions in PBS, and subsequently three 10 µL spots of each dilution were pipetted onto the surface of a TSA agar plate. The spots were allowed to dry for 20 min at room temperature before incubation at 37 °C for 18 h. The number of Colony Forming Units per mL (CFU/mL) was calculated as follows: Where d = dilution factor, and V = volume of inoculum (mL).

Detecting Alkaline Phosphatase in Planktonic Bacteria
Optically-adjusted bacterial cultures (1.4) underwent centrifugation (4000 g, 10 min) and were resuspended in an equal volume of 10 µM TCF-ALP in 50 mM Tris-HCl (pH 9.2). Alternatively, for TCF-ALP hydrogel analysis, 2 mL of the bacterial culture in 50 mM Tris HCl (pH 9.2) was transferred to a 12-well microtiter plate containing a 100 µM TCF-ALP based PVA hydrogel. These suspensions were subsequently protected from light and incubated at 32 °C for 24 h, unless stated otherwise. For both assays, 1 mL was removed from each suspension and centrifuged at 10 000 g for 3 min. The supernatant was subsequently placed into a black or clear 96-well microtiter plate for fluorescence and UV-Vis analysis, respectively. The fluorescence was measured using CLARIOstar fluorimeter, λ ex = 542 nm, λ em = 606 nm, and the UV-Vis by SPECTROstar Omega. Appropriate controls were carried out in tandem and a minimum of three biological replicates per bacterial strain were used.

ALP Activity of 96-well Biofilms
Overnight cultures of S. aureus NCTC 10788 were sub-cultured into fresh Mueller Hinton broth to attain a concentration of 10 6 CFU/mL, before 200 µL aliquots were placed into a 96-well microtiter plate and incubated at 32 °C for 24 h. After incubation, planktonic bacteria were discarded, and the remaining biofilm was washed three times with sterile 50 mM Tris HCl (pH 9.2). Plates were left to dry at room temperature for 20 min, before subsequent addition of 220µL of 10 µM TCF-ALP in 50 mM Tris-HCl (pH 9.2) and further incubation at 32 °C for 24 h. From each well, a 200 µL aliquot was removed and centrifuged at 10 000 g for 3 min. The supernatant was subsequently placed into a black 96-well microtiter plate for fluorescence analysis of ALP production.

ALP activity of bacterial isolates using p-nitrophenyl phosphate
Prior to the determination of enzymatic activity, a calibration curve of p-nitrophenol (p-NP) was obtained by measuring the absorbance at 405 nm of increasing concentrations of p-NP (0 -90 µM) in 50 mM Tris-HCl (pH 9.2) containing 250 mM NaOH.
Bacterial isolates (10 8 CFU/mL, 1 mL) were centrifuged (10,000g, 3 min) and resuspended in 950 µL of 10 mM p-nitrophenyl phosphate (p-NPP) in 50 mM Tris-HCl, pH = 9.2. After incubation at 32°C for 1 h, 50 µL of 5 M NaOH was added to stop the reaction. The resultant solution was centrifuged (10,000g, 3 min) and the absorbance of the supernatant was measured at 405 nm. The enzymatic activity of the solution was then determined by using the calibration curve of p-nitrophenol. 1 U (μmol/min) was defined as the amount of the enzyme that catalysed the conversion of one micromole of p-NPP per minute under the specified conditions of the assay method.

Role of Phosphatase Inhibitor on 96-well Biofilms
A 50 mM stock of sodium orthovanadate was prepared in deionised water (dH 2 O). Once dissolved, the pH was adjusted to 9.2 with NaOH and the resultant yellow solution (indicative of dimers) was boiled until colourless. Upon cooling, the pH was re-measured and adjusted if needed. This was repeated until the solution remained colourless.

Minimum Inhibitory Concentration (MIC)
The

Minimum Biofilm Inhibitory Concentration (MBIC)
One hundred microliter aliquots of sodium orthovanadate (50 mM in dH 2 O, pH 9.2) were added to a 96 well microtiter plate and serially diluted two-fold in Müeller Hinton broth. Next, 100 µL of S. aureus NCTC 10788 (10 6 CFU/mL in Mueller Hinton broth) was added to all relevant wells. The microtiter plate was then incubated at 32 °C for 18 h. After incubation, planktonic bacteria were discarded, and the remaining biofilm was washed three times with sterile 50 mM Tris HCl (pH 9.2) and left to dry for 20 min at room temperature. After, 220 µL of 0.1% Crystal Violet (CV) was added to all relevant wells, and the microtiter plate was incubated at room temperature for 30 min. After, the stain was removed, and the plate washed a further three times with sterile 50 mM Tris HCl (pH 9.2) before being left to dry for 3 h at room temperature. Next, 220 µL of 33 % acetic acid was added to the CV-stained biofilms and allowed to incubate for 15 minutes at room temperature, before 100 µL was transferred to a new microtiter plate and the optical density read at 590 nm. The MBIC was defined as the concentration which resulted in no detectable biofilm biomass.

Inhibition of ALP Activity
Overnight cultures of S. aureus NCTC 10788 were sub-cultured into fresh Mueller Hinton broth (10 6 CFU/mL) and placed into a 96-well microtiter plate before being incubated at 32 °C for 24 h. After incubation, planktonic bacteria were discarded, and the remaining biofilm was washed three times with sterile 50 mM Tris HCl (pH 9.2) and left to dry for 20 min at room temperature. Next, wells were pre-S9 treated with various concentrations of sodium orthovanadate (0 -3.75 mM; pH 9.2) for 30 min at room temperature, before addition of 10 µM TCF-ALP in 50 mM Tris-HCl (pH 9.2). Bacterial biofilms were then protected from light and incubated at 32 °C for a further 24 h. From each well, a 200 µL aliquot was removed and centrifuged at 10 000 g for 3 min. The supernatant was subsequently placed into a black 96-well microtiter plate for fluorescence analysis of ALP production.

Colony Biofilm Model
Colony biofilms were prepared as outlined in Thet et al. [25] with some modifications. First, 19 mm polycarbonate membranes were UV sterilised for 10 min on Mueller Hinton Agar, before being inoculated with 30 µL Artificial Wound Fluid (AWF; 50% fetal bovine serum in 50% peptone water

Sterilisation of Ex-vivo Porcine Skin Model
Porcine skin was washed with H 2 O before being cut into 2 x 2 cm squares, and subsequently underwent three 15-minute vortex cycles of washing in sterile dH 2 O. After, porcine skin was vortexed once for 15 min in 70 % ethanol, before a further two washes with sterile dH 2 O. Finally, the skin was UV-irradiated using a commercial UV source (Hamanatsu, Japan) equipped with a 254 nm UV lamp for 10 min before use.

ALP Activity
A 10 µL aliquot of S. aureus NCTC 10788 (10 8 CFU/mL in 50 mM Tris HCl pH 9.2) was added to the sterilised skin and left to dry for 20 min at room temperature. Next, 1 mL of 10 µM TCF-ALP in 50 S10 mM Tris-HCl (pH 9.2) was added to the skin via use of a Franz Cell. Inoculated porcine skin was then protected from light and incubated at 32 °C for 24 h. After, the suspension was removed and centrifuged at 10 000 g for 3 min. The supernatant was subsequently placed into a black 96-well microtiter plate for fluorescence analysis of ALP production. For TCF-ALP hydrogel analysis, a 100 µM TCF-ALP based PVA hydrogel was subsequently placed on the top of the inoculated skin. After being protected from light and incubated for 24 h at 32 °C, the hydrogels were removed from the skin and directly measured for fluorescence intensity.

Limit of Detection
Fluorescence intensity were measured for bacterial concentrations ranging from 10 5 -10 10 CFU/mL.
The fluorescence intensities were normalised and plotted against a Log CFU/mL scale. The linear portion of the graph was obtained, and the Limit of Detection (LOD) was defined as the x-intercept.   Cell counts were performed for bacterial strains that were unable to elicit a fluorescence response upon incubation with TCF-ALP. Figure S8 shows that the bacterial concentration of all E. coli strains (NCTC 10418, NSM59, and DH5α) were unaffected after 24 h incubation with TCF-ALP. All P.

W avelength / nm
aeruginosa strains (PAO1, P260, P885) had a statistically significant reduction in bacterial concentration; however, this correlated to a 1-log reduction, which should have a minimal effect on the efficacy of TCF-ALP. Therefore, other reasons must explain why E. coli and P. aeruginosa failed to produce a 'turn on' of TCF-ALP.