Highly Sensitive Detection of Bacteria by Binder-Coupled Multifunctional Polymeric Dyes

Infectious diseases caused by pathogens are a health burden, but traditional pathogen identification methods are complex and time-consuming. In this work, we have developed well-defined, multifunctional copolymers with rhodamine B dye synthesized by atom transfer radical polymerization (ATRP) using fully oxygen-tolerant photoredox/copper dual catalysis. ATRP enabled the efficient synthesis of copolymers with multiple fluorescent dyes from a biotin-functionalized initiator. Biotinylated dye copolymers were conjugated to antibody (Ab) or cell-wall binding domain (CBD), resulting in a highly fluorescent polymeric dye-binder complex. We showed that the unique combination of multifunctional polymeric dyes and strain-specific Ab or CBD exhibited both enhanced fluorescence and target selectivity for bioimaging of Staphylococcus aureus by flow cytometry and confocal microscopy. The ATRP-derived polymeric dyes have the potential as biosensors for the detection of target DNA, protein, or bacteria, as well as bioimaging.


Introduction
Infectious diseases caused by pathogens such as bacteria, viruses, and fungi remain a great burden on humanity [1]. As an example, antimicrobial resistance (AMR) is a major threat to human health. AMR-related infections have killed as many people as AIDS (acquired immunodeficiency syndrome) or malaria [2]. Under these circumstances, target-specific identification of pathogens is critical for effective medical intervention and decontamination of the infected areas [3]. Colony counting, immunological, and polymerase chain reaction (PCR) techniques are the traditional methods for pathogen identification [4][5][6][7]. However, these methods require time-consuming and complicated procedures such as cell culture, antigen/antibody treatment, and cell lysis/DNA amplification [5][6][7]. In this context, fluorescent labeling and detection have emerged as promising tools for pathogen visualization and identification, due to their simple labeling procedure, sensitivity, and stability [3,[8][9][10]. Moreover, conjugation of fluorescent materials with a biological binder such as an antibody [11,12], an aptamer [13,14], or the cell-wall binding domain of a lytic enzyme (CBD) [15][16][17] allows for the targeting of a specific pathogen.
Various classes of fluorescent materials such as small-molecule organic dyes [18][19][20][21], fluorescent proteins [22][23][24], self-fluorescent polymers [10,[25][26][27], dye-labeled polymeric nanoparticles [28][29][30][31][32][33][34], and quantum dots [35][36][37][38] have been explored. Among these, fluorescent dyes have gained popularity due to their commercial availability, ease of operation, and high resolution [39]. This has paved the way for the development of fluorescent dye Herein, we report the synthesis and characterization of a polymeric dye complex containing a binder such as an antibody or the cell-wall binding domain (CBD) from a lytic enzyme for a highly sensitive bioimaging technique for pathogen identification. Putative autolysin from Staphylococcus aureus (SA1) contains a cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain and a putative CBD [118]. The CBD from SA1 was successfully expressed and exhibited high selectivity to Staphylococcus aureus, similar to the CBD of lysostaphin, a lytic enzyme known for its specific targeting of Staphylococcus aureus [119]. The approach involves the coupling of Staphylococcus aureus targeting polyclonal antibody or CBD of SA1 to rhodamine dye-labeled copolymers comprised of oligo (ethylene oxide) methyl ether methacrylate or carboxy betaine methacrylate (CBMA). The polymers were grafted from a biotin-functionalized ATRP initiator under blue-light irradiation using Eosin-Y/Cu-mediated fully oxygen-tolerant ATRP technique. The nondestructive binding properties of the copolymeric dye complex were tested on the target bacterial cells and were applied to the bioimaging of target bacteria using fluorescence detection and confocal microscopy analysis.

Characterization of Biotinylated Copolymeric Rhodamine B by 1 H NMR Spectroscopy and Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS) Detectors
Before analysis, the synthesized biotinylated copolymeric dyes were purified by dialysis in deionized water using SpectraPor ® 10 kDA cutoff dialysis membrane for 48 h and then lyophilized. 1 H NMR spectra were recorded on Bruker Avance III 500 MHz spectrometers with D 2 O as the solvent. SEC-MALS measurements were performed using the Agilent SEC system (Agilent, 1260 Infinity II with UV detector) coupled with MALS, DLS, Viscometer, and RI detectors (Wyatt Technology Corporation, Santa Barbara, CA, USA). Measurements were performed using the Waters Ultrahydrogel Linear column with 1X DPBS as an eluent at room temperature and a flow rate of 0.5 mL/min.

Kinetics of Photoinduced ATRP
The ATRP reaction mixture (5 mL) was prepared according to the general procedure described above for the synthesis of BT-p(OEOMA 500 -RDMA (4)). The final concentrations were OEOMA 500 (300 mM), RDMA were added to a 1-dram (12/96 mm) vial equipped with a magnetic stir bar. The polymerization mixture in an uncapped vial was stirred at 500 rpm for 40 min under green LEDs (520 nm, 9.0 mW/cm 2 ). The samples were taken at regular intervals, quenched with 1,4-bis(3-isocyanopropyl) piperazine [120], and then analyzed by 1 H NMR and SEC.

Photostability Assessment of Biotinylated Copolymeric Rhodamine B
The photostability of the biotinylated copolymeric dyes was accessed by measuring the fluorescence readout every 30 min for 18 h. The experiment was carried out overnight using a BioTek Synergy H1 microplate reader. The copolymer dye was dissolved in 1X PBS buffer to reach a final concentration of 60 µM and placed in a 96-well polystyrene black-bottom plate. Triplicate samples of the same concentration were measured with a control sample containing only the buffer. Mineral oil (50 µL) was added to each well to prevent evaporation. Samples were incubated in a plate reader (xenon flash lamp, high energy) at 37 • C in the measurement chamber. The fluorescence intensity was measured by top optics using the monochromator filter set: excitation at 540 nm and emission at 570 nm, every 30 min.

Preparation of Cell-Wall Binding Domain (CBD)
The DNA sequence of the cell-wall binding domain of SA1 [119] including an avi-tag and BirA (biotin ligase) was subcloned into a pGS-21a and pCDF-duet vector between NdeI and XhoI, respectively. BL21(DE3) competent cells were then cotransformed with pGS-21a with SA1BD gene and pCDF-duet with birA gene. One milliliter of the saturated overnight culture was inoculated into 100 mL of fresh LB media containing ampicillin (100 µg/mL) and grown until the absorbance at 600 nm reaches approximately 0.4. IPTG and D-biotin were added to a final concentration of 1 mM and 50 µM, respectively, and cells were cultured at 16 • C and 150 rpm overnight. Afterward, cells were pelleted by centrifuging (4000 rpm) at 4 • C for 15 min and resuspended in 10 mL of cell lysis buffer in native purification buffer (NPB, 20 mM sodium phosphate, and 500 mM NaCl, pH 8.0) containing PMSF (1 mM), lysozyme (100 µg/mL), bovine pancrease Dnase I (100 µg/mL), and glycerol (5%, v/v). The cell suspensions were sonicated in ice using Misonix Sonicator ® 3000 (Farmingdale, NY, USA) for 30 min with 1 s pulses and then centrifuged at 4000 rpm for 15 min to collect the supernatant. The His-tagged protein in the supernatant was purified using nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography. The bound protein was washed once with NPB containing PMSF (1 mM) and five times with NPB containing imidazole (20 mM). The protein was eluted with an elution buffer (NPB containing imidazole (200 mM)) and was dialyzed against PBS at pH 7.4 using an 8 kDa molecular weight cutoff membrane (SpectrumLabs, Arden, NC, USA). Protein concentrations were determined spectrophotometrically at 280 nm using a NanoDrop ND-1000 (ThermoFisher, Waltham, MA, USA).

Construction and Characterization of Antibody/CBD-Copolymeric Rhodamine B Complex
To construct the antibody/CBD complexes with rhodamine B dye, biotinylated polyclonal antibody or CBD solution was first mixed with NeutrAvidin in phosphate-buffered saline (PBS, pH 7.4) and incubated at room temperature for 30 min. Biotinylated monomeric or copolymeric rhodamine B solution was then added to the mixture, followed by incubation at room temperature for 30 min. The molar ratio of biotinylated antibody/CBD, NeutrAvidin, and monomeric/copolymeric rhodamine B was 1:1:1.
Fluorescence from the prepared complexes in the presence of target bacteria were measured to determine the binding. Briefly, 30 µL of the saturated overnight culture were inoculated into 3 mL of fresh LB media and grown until the absorbance at 600 nm reached approximately 0.4. Afterward, cells were centrifuged and washed three times with PBS (pH 7.4). Antibody/CBD complex fusion proteins were added to the target cells (5 × 10 8 cells/mL) and incubated at room temperature for 15 min. After incubation, the resulting mixtures were washed three times with PBS with 0.2% Tween20 to remove the unbound complex and adjusted to 5 × 10 8 cells/mL. The fluorescence intensity of the mixture was then measured using a microplate reader (SpectraMax M5, San Jose, CA, USA) (λ ex = 546 nm and λ em = 580 nm). The fluorescence from complexes at the surface of bacteria was also measured using flow cytometry. Mixtures were diluted 10-fold in PBS to 10 7 cells/mL prior to flow cytometry. Flow cytometry was performed using the BD LSRII flow cytometer (BD Biosciences, Franklin Lake, NJ, USA) with 20,000 events collected for each sample. Gating and further flow cytometry analysis were performed using FlowJo.

Confocal Laser Scanning Microscopy (CLSM)
Antibody/CBD complex fusion proteins were added to bacterial cells (5 × 10 8 cells/mL) and incubated at room temperature for 15 min. After incubation, the resulting mixtures were washed three times with PBS with 0.2% Tween20 to remove the unbound complex and adjusted to 5 × 10 8 cells/mL. A PFA solution (4%) was then added to the resulting mixtures to fix the bacteria cells. After incubation on ice for 30 min, the mixture was washed twice with PBS. Each 10 µL aliquot of the prepared mixed suspensions was added to clean glass slides and lightly covered using coverslips. The samples were excited at 546 nm, and the emission was recorded between 556 and 632 nm. The samples were examined by confocal laser scanning microscopy (LSM780) with a 93× glycerol immersion objective lens using a 546 nm laser (Carl Zeiss A.G., Oberkochen, Germany). Microscopy images were prepared and analyzed using ImageJ (Version 1.53k, National Institutes of Health, Bethesda, MD, USA). Briefly, raw TIF images for each microscopy sample were imported into ImageJ. For each image, the fluorescence was measured for 25 cells, and 5 background spots were measured for correction. The size of measurement for each cell and background was kept consistent within images. The corrected total cell fluorescence (CTCF) for each cell was calculated. Confocal microscopy was also used to examine the specificity of the antibody/CBD complex fusion proteins by using a mixture of both S. aureus and B. anthracis cells. The same procedure as above was used where the only modification is using a 1:1 mixture of S. aureus and B. anthracis cells. Confocal microscopy was performed using the same parameters above. Brightfield images for bacterial mixtures were also taken.

Synthesis and Characterization of Biotinylated Copolymeric Rhodamine B
The synthesis of biotinylated multifunctional dye copolymers was performed using a recently developed fully oxygen-tolerant, photoinduced atom transfer radical polymerization (ATRP) [108]. The copolymerization of zwitterionic and hydrophilic CBMA monomer and dye-labeled rhodamine-B-methacrylate (RDMA) monomer was performed in an aqueous medium under blue-light irradiation (λ max = 450 nm, 25.0 mW/cm 2 ), using biotin-I as the initiator, Eosin Y (EYH 2 ) as the organic photoredox catalyst, and CuBr 2 /TPMA as the deactivator (Figure 1). mixtures to fix the bacteria cells. After incubation on ice for 30 min, the mixture was washed twice with PBS. Each 10 µL aliquot of the prepared mixed suspensions was added to clean glass slides and lightly covered using coverslips. The samples were excited at 546 nm, and the emission was recorded between 556 and 632 nm. The samples were examined by confocal laser scanning microscopy (LSM780) with a 93× glycerol immersion objective lens using a 546 nm laser (Carl Zeiss A.G., Oberkochen, Germany). Microscopy images were prepared and analyzed using ImageJ (Version 1.53k, National Institutes of Health, Bethesda, MD, USA). Briefly, raw TIF images for each microscopy sample were imported into ImageJ. For each image, the fluorescence was measured for 25 cells, and 5 background spots were measured for correction. The size of measurement for each cell and background was kept consistent within images. The corrected total cell fluorescence (CTCF) for each cell was calculated. Confocal microscopy was also used to examine the specificity of the antibody/CBD complex fusion proteins by using a mixture of both S. aureus and B. anthracis cells. The same procedure as above was used where the only modification is using a 1:1 mixture of S. aureus and B. anthracis cells. Confocal microscopy was performed using the same parameters above. Brightfield images for bacterial mixtures were also taken.

Synthesis and Characterization of Biotinylated Copolymeric Rhodamine B
The synthesis of biotinylated multifunctional dye copolymers was performed using a recently developed fully oxygen-tolerant, photoinduced atom transfer radical polymerization (ATRP) [108]. The copolymerization of zwitterionic and hydrophilic CBMA monomer and dye-labeled rhodamine-B-methacrylate (RDMA) monomer was performed in an aqueous medium under blue-light irradiation (λmax = 450 nm, 25.0 mW/cm 2 ), using biotin-I as the initiator, Eosin Y (EYH2) as the organic photoredox catalyst, and CuBr2/TPMA as the deactivator (Figure 1). Similarly, amphiphilic copolymers comprised of PEG backbone were also synthesized by copolymerization of oligo (ethylene oxide) methyl ether methacrylate (OEOMA 500 ) monomer with RDMA. 1 H NMR was analyzed to compute conversion of monomers; the peak integral from 4.26 to 4.32 ppm corresponding to the monomer was set at 100 at t = 0, and the broad peak between 4.05 and 4.20 ppm corresponding to the polymer was observed at t = 60 min. Within 60 min, the CBMA and OEOMA 500, and RDMA reached a high conversion (≈80%), which was used to compute theoretical molecular weight (M n,th ). 1 H NMR spectra of the purified polymer samples was also recorded (Figures S1 and S2). The purified polymer samples were then analyzed by the SEC-MALS technique (Table 1), where a good agreement between M n,th and the observed absolute molecular weight (M n, abs ) revealed well-controlled polymerizations (Figure 2a).   Table 1 after 60 min. Copolymerization kinetics of the optimized EYH2/Cu-catalyzed ATRP for the synthesis of BT-p(OE-OMA500-RDMA (4)). (b) First-order kinetic plot. (c) Evolution of molecular weight and molecular weight distribution with conversion (black dots represent molecular weight and pink dots represent molecular weight distribution).

Characterization of Biotinylated Polymeric Dyes and Their Complexes with Selective Binders
We complexed the biotinylated polymeric dyes with NeutrAvidin and a cell binder such as biotinylated S. aureus polyclonal antibody or biotinylated CBD using biotin/Neu-trAvidin interactions and tested their binding toward the target S. aureus cells using fluorescence detection (Figure 3). For fluorescence measurements of antibody/CBD-polymeric dye complexes on the surface of S. aureus, we used an excitation and emission at 546 nm and 580 nm, respectively, based on the fluorescent spectra of p(OEOMA500-RDMA (4)) ( Figure S4). In both antibody and CBD cases, the p(OEOMA500-RDMA (4)) complex showed the best performance without any background fluorescence (Figure 4a) compared to p(OEOMA500-RDMA (2)) and p(CBMA-RDMA (2)) complexes. The result implies that a higher molar ratio of rhodamine B dye was incorporated in the p(OEOMA500-RDMA (4)) backbone than in the p(OEOMA500-RDMA (2)) backbone. Also, in the case of zwitterionic BT-p(CBMA-RDMA (2)), we observed a background fluorescence, which can be attributed to the distinct characteristics of the copolymer backbone, contributing to its zwitterionic and hydrophilic nature. This unique feature of the copolymer backbone may lead to nonspecific interactions to zwitterionic teichoic acid polymers located within the Grampositive cell wall. [121,122], 1 (resulting in high background fluorescence. In contrast, both p(OEOMA500-RDMA (2)) and p(OEOMA500-RDMA (4)) complexes with PEGylated side chains can effectively minimize the nonspecific interactions with the cell surface. In all cases, the antibody-induced fluorescence was higher than the CBD-induced fluorescence. This may be because the number of binding sites for the polyclonal antibody is higher than that of CBD at the upper cell wall structure of the Gram-positive S. aureus. The con-  (Figures 2b and S3), and exhibited first-order kinetics. The monomer conversion determined via 1 H NMR revealed statistical incorporation of the fluorescent monomer (RDMA) within the polymer chain. A good agreement between theoretical and experimental molecular weights was observed. In addition, SEC traces revealed that molecular weights increased as a function of monomer conversion, and dispersity values remained low Ð < 1.3). The ATRP technique enabled efficient and rapid synthesis of copolymer chains without the need for deoxygenation, with the desired degree of polymerization, desired molar ratio of RDMA, predictable molecular weights, narrow molecular weight distribution, and homogenously distributed fluorescent dye monomers (Figure 2c).

Characterization of Biotinylated Polymeric Dyes and Their Complexes with Selective Binders
We complexed the biotinylated polymeric dyes with NeutrAvidin and a cell binder such as biotinylated S. aureus polyclonal antibody or biotinylated CBD using biotin/NeutrAvidin interactions and tested their binding toward the target S. aureus cells using fluorescence detection (Figure 3). For fluorescence measurements of antibody/CBD-polymeric dye complexes on the surface of S. aureus, we used an excitation and emission at 546 nm and 580 nm, respectively, based on the fluorescent spectra of p(OEOMA500-RDMA (4)) ( Figure S4). In both antibody and CBD cases, the p(OEOMA 500 -RDMA (4)) complex showed the best performance without any background fluorescence (Figure 4a) compared to p(OEOMA 500 -RDMA (2)) and p(CBMA-RDMA (2)) complexes. The result implies that a higher molar ratio of rhodamine B dye was incorporated in the p(OEOMA 500 -RDMA (4)) backbone than in the p(OEOMA 500 -RDMA (2)) backbone. Also, in the case of zwitterionic BT-p(CBMA-RDMA (2)), we observed a background fluorescence, which can be attributed to the distinct characteristics of the copolymer backbone, contributing to its zwitterionic and hydrophilic nature. This unique feature of the copolymer backbone may lead to nonspecific interactions to zwitterionic teichoic acid polymers located within the Grampositive cell wall. [121,122], 1 (resulting in high background fluorescence. In contrast, both p(OEOMA 500 -RDMA (2)) and p(OEOMA 500 -RDMA (4)) complexes with PEGylated side chains can effectively minimize the nonspecific interactions with the cell surface. In all cases, the antibody-induced fluorescence was higher than the CBD-induced fluorescence. This may be because the number of binding sites for the polyclonal antibody is higher than that of CBD at the upper cell wall structure of the Gram-positive S. aureus. The concentration of p(OEOMA 500 -RDMA (4)) species in the complexation process was optimized and fixed at 6 µM ( Figure S5). To assess the stability of biotinylated copolymer rhodamine B, the fluorescence intensity of the synthesized copolymers was monitored overnight ([BT-p(OEOMA-RDMA (4)] = 60 µM). The polymer sample was incubated in 1X PBS buffer in a plate reader at 37 °C in the measurement chamber for 18 h. The negligible change in the fluorescence  We then assessed binding onto single bacterial cells using flow cytometry. The addition of each binder-p(OEOMA500-RDMA (4)) complex generated a clear change in the fluorescence intensity compared with the target S. aureus cells alone ( Figure 5), suggesting that the fluorescence of each binder-p(OEOMA500-RDMA (4)) complex comes from binding to the surface of S. aureus cells. Furthermore, we confirmed the specificity of these complexes using flow cytometry. In both binder cases, the complex did not bind to B. anthracis Sterne cells, as no fluorescence was detected. These results suggest that we have generated complexes that are target-specific and can bind to the target bacterium, induced by antibody or CBD.

Antibody/CBD-Polymeric Dyes Complex for Bioimaging Application
We applied the signal-enhancing property of each binder-p(OEOMA500-RDMA (4)) complex for the bioimaging of target bacteria. When we prepared S. aureus cells with binder-p(OEOMA500-RDMA (4)) complex, all the target bacterial cells with binder-p(OE-OMA500-RDMA (4)) complex showed red emission in each image (Figure 6a-d). In addition, the fluorescence images showed the same trends that we had previously obtained using fluorescence detection on a plate reader. Further analysis of these images was performed to quantify the fluorescence from the single cell by corrected total cell fluorescence (CTCF) analysis using the ImageJ program. The CTCF of antibody-and CBD-p(OE-OMA500-RDMA (4)) complexes were 1.6 and 3.6 times higher than that of antibody-and CBD-RD complexes, respectively (Figure 6e). Additionally, in a mixture of cells, under confocal microscopy, we specifically distinguished the presence of the target bacteria from To assess the stability of biotinylated copolymer rhodamine B, the fluorescence intensity of the synthesized copolymers was monitored overnight ([BT-p(OEOMA-RDMA (4)] = 60 µM). The polymer sample was incubated in 1X PBS buffer in a plate reader at 37 • C in the measurement chamber for 18 h. The negligible change in the fluorescence intensity of the polymer samples during the experiment indicated the high stability of the polymeric dyes under these experimental conditions ( Figure S6).
Next, the fluorescence of antibody/CBD-p(OEOMA 500 -RDMA (4)) complexes was measured and compared with biotinylated monomeric rhodamine B complexes with Neu-trAvidin and antibody/CBD (antibody/CBD-RD complexes) (Figure 3). The fluorescence of the antibody-and CBD-p(OEOMA 500 -RDMA (4)) complexes were 2.6 and 3.7 times higher than that of the antibody-and CBD-RD complexes (Figure 4b), respectively, suggesting that the signal of each binder-p(OEOMA 500 -RDMA (4)) complex was improved by increasing the number of rhodamine B dyes on the copolymer without background fluorescence from nonspecific binding of the copolymer.
We then assessed binding onto single bacterial cells using flow cytometry. The addition of each binder-p(OEOMA 500 -RDMA (4)) complex generated a clear change in the fluorescence intensity compared with the target S. aureus cells alone ( Figure 5), suggesting that the fluorescence of each binder-p(OEOMA 500 -RDMA (4)) complex comes from binding to the surface of S. aureus cells. Furthermore, we confirmed the specificity of these complexes using flow cytometry. In both binder cases, the complex did not bind to B. anthracis Sterne cells, as no fluorescence was detected. These results suggest that we have generated complexes that are target-specific and can bind to the target bacterium, induced by antibody or CBD.  We then assessed binding onto single bacterial cells using flow cytometry. The addition of each binder-p(OEOMA500-RDMA (4)) complex generated a clear change in the fluorescence intensity compared with the target S. aureus cells alone ( Figure 5), suggesting that the fluorescence of each binder-p(OEOMA500-RDMA (4)) complex comes from binding to the surface of S. aureus cells. Furthermore, we confirmed the specificity of these complexes using flow cytometry. In both binder cases, the complex did not bind to B. anthracis Sterne cells, as no fluorescence was detected. These results suggest that we have generated complexes that are target-specific and can bind to the target bacterium, induced by antibody or CBD.

Antibody/CBD-Polymeric Dyes Complex for Bioimaging Application
We applied the signal-enhancing property of each binder-p(OEOMA500-RDMA (4)) complex for the bioimaging of target bacteria. When we prepared S. aureus cells with binder-p(OEOMA500-RDMA (4)) complex, all the target bacterial cells with binder-p(OE-

Antibody/CBD-Polymeric Dyes Complex for Bioimaging Application
We applied the signal-enhancing property of each binder-p(OEOMA 500 -RDMA (4)) complex for the bioimaging of target bacteria. When we prepared S. aureus cells with binderp(OEOMA 500 -RDMA (4)) complex, all the target bacterial cells with binder-p(OEOMA 500 -RDMA (4)) complex showed red emission in each image (Figure 6a-d). In addition, the fluorescence images showed the same trends that we had previously obtained using fluorescence detection on a plate reader. Further analysis of these images was performed to quantify the fluorescence from the single cell by corrected total cell fluorescence (CTCF) analysis using the ImageJ program. The CTCF of antibody-and CBD-p(OEOMA 500 -RDMA (4)) complexes were 1.6 and 3.6 times higher than that of antibody-and CBD-RD complexes, respectively (Figure 6e). Additionally, in a mixture of cells, under confocal microscopy, we specifically distinguished the presence of the target bacteria from the nontarget using these complexes (Figure 6f,g). These results suggest that these binder-p(OEOMA 500 -RDMA (4)) complexes can be applied for bioimaging to visualize specific bacteria. , x FOR PEER REVIEW 11 of 17 the nontarget using these complexes (Figure 6f,g). These results suggest that these binderp(OEOMA500-RDMA (4)) complexes can be applied for bioimaging to visualize specific bacteria.

Conclusions
We have developed ATRP-derived copolymeric multifunctional rhodamine B dyes and attached them to binders such as an antibody or CBD for selective binding of target bacterial cells. The photoredox/Cu-catalyzed ATRP technique enabled the efficient and rapid synthesis of well-defined copolymers with multiple fluorescent dyes. Antibody/CBD-polymeric dye complex showed both enhanced fluorescence and target selectivity for bioimaging. This is due to the special structural property of this complex, consisting of multiple fluorescent dyes and a single binding molecule. The combination of this unique property of the polymeric dye and binder-induced targeting can also be applied to conjugate multiple signaling molecules such as quantum dots, DNA, and enzymes, followed by the potential applications in pathogen detection, and selective microbial decontamination, as well as bioimaging. The present work has also opened the potential application of ATRP-derived polymeric dyes in biosensors for detection of the DNA or protein biomarkers.

Conclusions
We have developed ATRP-derived copolymeric multifunctional rhodamine B dyes and attached them to binders such as an antibody or CBD for selective binding of target bacterial cells. The photoredox/Cu-catalyzed ATRP technique enabled the efficient and rapid synthesis of well-defined copolymers with multiple fluorescent dyes. Antibody/CBDpolymeric dye complex showed both enhanced fluorescence and target selectivity for bioimaging. This is due to the special structural property of this complex, consisting of multiple fluorescent dyes and a single binding molecule. The combination of this unique property of the polymeric dye and binder-induced targeting can also be applied to conjugate multiple signaling molecules such as quantum dots, DNA, and enzymes, followed by the potential applications in pathogen detection, and selective microbial decontamination, as well as bioimaging. The present work has also opened the potential application of ATRPderived polymeric dyes in biosensors for detection of the DNA or protein biomarkers.