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Qicheng Zhang, Si Chen, Xiaoting Xue, [Solmaz Hajizadeh](https://orcid.org/0000-0002-0348-8756), [Tomohiko Yamazaki](https://orcid.org/0000-0003-2136-8042), [Lei Ye](https://orcid.org/0000-0002-3646-4072)

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[Cationic Polymer Brushes Functionalized with Carbon Dots and Boronic Acids for Bacterial Detection and Inactivation](https://mdr.nims.go.jp/datasets/90f883bf-bc73-4e82-bd63-4bc8c40b040f)

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Cationic Polymer Brushes Functionalized with Carbon Dots and Boronic Acids for Bacterial Detection and InactivationCationic Polymer Brushes Functionalized with Carbon Dots andBoronic Acids for Bacterial Detection and InactivationQicheng Zhang, Si Chen, Xiaoting Xue, Solmaz Hajizadeh, Tomohiko Yamazaki, and Lei Ye*Cite This: ACS Omega 2025, 10, 14536−14546 Read OnlineACCESS Metrics & More Article Recommendations *sı Supporting InformationABSTRACT: Drug-resistant bacterial infections are among the most severe physiological challenges facing human health.Therefore, the detection and inactivation of pathogenic bacteria remains a crucial therapeutic goal in modern society. In this study,we design multifunctional nanocomposites aimed at bacterial binding, fluorescence labeling, and synergistic antibacterial treatment.These nanocomposites are prepared by introducing cationic polymers with quaternary ammonium compounds onto silicananoparticles using surface-initiated atom transfer radical polymerization, followed by incorporation of copper-doped carbon dotsand modification of boronic acid. The cationic polymer units and boronic acid end groups enhance the bacterial binding capacityand synergistic bactericidal effects in cooperation with the carbon dots. Due to the stable fluorescent properties of carbon dots, thenanocomposites can generate fluorescence signals around bacteria, enabling bacterial fluorescence imaging. Overall, this studydemonstrates a multifunctional nanocomposite-assisted strategy for bacterial labeling, imaging, and deactivation, providing a novelapproach for bacterial detection and synergistic treatment.1. INTRODUCTIONBacteria-induced infections are among the most severe medicalproblems, causing many serious diseases such as plague, sepsis,and infective endocarditis.1,2 The spread of such infections canalso result in substantial economic losses in poultry productionand food industry, increasingly attracting public attentionworldwide.3,4 Naturally, the detection and deactivation ofpathogenic bacteria are essential for responding to bacterialthreats. However, this remains a significant challenge and apractical issue of great importance.Antibiotic therapies have been widely used to combatmicrobial infections, but many pathogens have evolved drugresistance due to the overuse of these antibacterial agents.5−7Therefore, new approaches to prevent the emergence ofresistant bacteria, such as cationic compounds (or polymers)with positive charges, photothermal materials that release heatenergy, and inhibitors that disrupt microbial quorum sensing,have been widely explored.8−10 There are several alternativetreatments that destroy pathogens by generating reactiveoxygen species under different trigger conditions, such as lasers(photodynamic therapy) and certain transition metal elements(chemodynamic therapy).11,12 Additionally, antimicrobialpeptides, which naturally form polypeptide sequencescomposed of cationic and hydrophobic amino acids withdirect antibacterial activity, are also powerful strategies toaddress the impending crisis of antimicrobial resistance.13Another critical issue is the selection of analytical technologyfor the detection of bacteria. Compared with the time-consuming and labor-intensive bacterial colony-forming unitcounting method, fluorescence-based techniques offer low cost,fast response, and easy accessibility.14,15 Moreover, thedetection process of bacteria inevitably involves bacterialbinding. Boronate affinity techniques have emerged as apowerful tool in bioconjugation, molecular identification, andReceived: February 18, 2025Revised: March 16, 2025Accepted: March 26, 2025Published: April 2, 2025Articlehttp://pubs.acs.org/journal/acsodf© 2025 The Authors. Published byAmerican Chemical Society14536https://doi.org/10.1021/acsomega.5c01507ACS Omega 2025, 10, 14536−14546This article is licensed under CC-BY 4.0Downloaded via NATL INST FOR MATLS SCIENCE (NIMS) on September 27, 2025 at 04:46:25 (UTC).See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Qicheng+Zhang"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Si+Chen"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Xiaoting+Xue"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Solmaz+Hajizadeh"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Tomohiko+Yamazaki"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Lei+Ye"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/showCitFormats?doi=10.1021/acsomega.5c01507&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?goto=articleMetrics&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?goto=recommendations&?ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?goto=supporting-info&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=abs1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=abs1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=abs1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=abs1&ref=pdfhttps://pubs.acs.org/toc/acsodf/10/14?ref=pdfhttps://pubs.acs.org/toc/acsodf/10/14?ref=pdfhttps://pubs.acs.org/toc/acsodf/10/14?ref=pdfhttps://pubs.acs.org/toc/acsodf/10/14?ref=pdfhttp://pubs.acs.org/journal/acsodf?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://doi.org/10.1021/acsomega.5c01507?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://http://pubs.acs.org/journal/acsodf?ref=pdfhttps://http://pubs.acs.org/journal/acsodf?ref=pdfhttps://acsopenscience.org/researchers/open-access/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/chromatographic separation due to the formation of boronateester complexes between boronic acid and the cis-diolstructures of polysaccharides and oligosaccharides on thesurface of pathogens, which can help us avoid the use ofexpensive antibodies.16−19 Therefore, designing a method thatcan noninvasively and efficiently bind, detect, and inactivatebacteria using fluorescent detection and boronate affinity is ofgreat significance.20−22To achieve this goal, the strengths of various bacteria-relatedapproaches should be integrated into a single platform.Polymers are ideal nanoarchitectures for functionalizing andfabricating multipurpose materials. Polymers prepared usingcontrolled radical polymerization (CRP) methods can beefficiently designed, endowing them with well-defined blocksequences and desired molecular weights.23−26 Among variousCRP methods, atom transfer radical polymerization (ATRP) iswidely used to create new copolymer materials with diversefunctionalities and precisely controlled molecular weights.27,28When an initiator is prefixed onto a support, surface-initiatedatom transfer radical polymerization (SI-ATRP) can beconducted to conveniently graft various types of functionalizedpolymer brushes onto solid substrates, such as silica nano-particles, magnetic beads, metallic surfaces, and engineeredscaffolds.29−32 The solid substrates facilitate straightforwardpurification of the polymer products through simple filtrationor sedimentation. Typically, these polymers consist of twodistinct functional parts containing different chemical groupsand chain segments with different functions. For example,cationic monomers that carry quaternary ammonium com-pounds (QACs), such as [2-(methacryloyloxy)-ethyl]-trimethylammonium chloride (METAC), have emerged aspotential antibacterial building blocks to prepare the polymerbrushes.33,34 The cationic polymers inactivate bacteria throughstrong interactions with the cytoplasmic membrane andirreversible destruction of the bacterial membrane integrity.Glycidyl methacrylate (GMA) is an excellent tunablemonomer because its epoxy group can undergo a ring-openingreaction to enable conjugation of additional small moleculesand even biomacromolecules onto polymer chains.35,36Polymer coatings composed of QACs monomers and GMAcan be chemically modified to enhance antibacterial activityand add new functionalities.As a zero-dimensional carbon material with abundantsurface functional groups and excellent luminescence features,carbon quantum dots (CDs) have shown great potential inbiolabeling and antibacterial fields.37,38 Generally, the innercore of CDs is composed of sp2 hybridized carbon, and theouter shell has organic functional groups. CDs can be dopedwith various heteroatoms and metal atoms to enhance thedelocalization of electrons and the physicochemical propertiesof CDs.39 Compared to CDs doped with heteroatoms, metal-doped CDs exhibit higher surface charge and enhancedelectron transfer, damaging bacteria through charge-inducedphysical destruction and reactive oxygen species-triggeredoxidative stress.40,41 Simultaneously, the surface of CDs can bedesigned to have various functional groups by using differentScheme 1. Schematic Illustration for the Synthesis of the Multifunctional Nanocomposite Si@co@BA and Application of theMaterial for Binding, Detection, and Inactivation of BacteriaACS Omega http://pubs.acs.org/journal/acsodf Articlehttps://doi.org/10.1021/acsomega.5c01507ACS Omega 2025, 10, 14536−1454614537https://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=sch1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=sch1&ref=pdfhttp://pubs.acs.org/journal/acsodf?ref=pdfhttps://doi.org/10.1021/acsomega.5c01507?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asprecursors, enabling CDs to be further modified with specificmolecular recognition entities for versatile applications.42,43Furthermore, CDs demonstrate an appealing and stablefluorescence nature originating from luminescent conjugatedunits composed of isolated sp2 carbon clusters. By adjustingstructural differences, contributions from the carbon core andsurface states, and the presence of final fluorophores, thephotoluminescence of CDs can be effectively shifted to meetvarious bacterial imaging and detection needs.44−46This work designed a new nanohybrid material to providecellular binding, fluorescence labeling, and synergistic anti-bacterial activity by combining copolymer brushes grafted ontoamino-functionalized silica with copper-doped CDs (Scheme1). First, SI-ATRP was used to coat the poly(METAC-co-GMA) copolymer on silica, which endowed the nano-composites with QACs-mediated antibacterial activity andreactive sites for introducing other functional materials.Subsequently, copper-doped CDs containing amino groupswere linked to the copolymer chain through a ring-openingreaction of the epoxy groups (GMA) and amino groups(CDs), achieving a fluorescent capability and enhancedantibacterial activity. By immobilizing copolymers and CDsonto silica nanoparticles, the polymer chains and CDs can beeasily separated for further use and a longer retention time.Finally, the remaining amino groups could react with aldehydegroups on 4-formylphenylboronic acid (FPBA) to add boronicacid for binding bacteria. The bacterial binding was facilitatedby the multiple boronic acid ligands that form reversibleboronate ester bonds with the polysaccharides and oligosac-charides in microbial cells. The antibacterial activity,copolymer properties, and physicochemical characteristics ofthe nanocomposites were investigated. Fluorescence micros-copy, bacterial transmission electron microscopy (TEM), andflow cytometry were also used to demonstrate the labeling andimaging functions of the nanocomposites.2. EXPERIMENTAL SECTION2.1. Materials. Tetraethylorthosilicate (TEOS), ammo-nium hydroxide (25%), (3-aminopropyl)-triethoxysilane(APTES), triethylamine (TEA), 2-bromoisobutyryl bromide(BIBB), [2-(methacryloyloxy)ethyl]trimethylammoniumchloride solution (METAC, 75%), glycidyl methacrylate(GMA), N,N,N′,N″,N″-pentamethyldiethylenetriamine(PMEDTA), copper(II)bromide (CuBr2), L-ascorbic acid(AscA), citric acid, ethylenediamine, 4-formylphenyl-boronicacid (FPBA), sodium cyanoborohydride (NaBH3CN), meth-anol, toluene, tetrahydrofuran (THF), and alizarin red S(ARS) were purchased from Sigma-Aldrich. Copper disodiumethylenediaminetetraacetate (Na2[Cu(EDTA)]) was pur-chased from TCI (Tokyo, Japan). Yeast extract and agarwere purchased from Merck. Tryptone was purchased fromDuchefa Biochemie. Sodium chloride (NaCl), disodiumhydrogen phosphate (Na2HPO4), potassium dihydrogenphosphate (KH2PO4), and potassium chloride (KCl) werepurchased from Fisher Scientific. Lysogeny broth (LB) mediawere prepared by dissolving yeast extract (5 g/L), tryptone (10g/L), and NaCl (10 g/L) in water. Agar plates were preparedby adding extra agar (15 g/L) to the LB media.2.2. Synthesis of Initiator-Modified Silica Nano-particles (Si@BiBB). Silica nanoparticles (SiO2) wereprepared using a “one-step” Stöber reaction.47 Typically, 33mL of water, 100 mL of methanol, and 22.4 mL of ammoniumhydroxide (25%) were added into a 500 mL round-bottomflask and agitated at room temperature. Subsequently, amixture of 130 mL of methanol containing 13.8 mL of TEOSwas quickly added to the solution, which was then stirred for 8h. The silica nanoparticles were separated by centrifugation at10,000 rpm for 10 min and washed several times with waterand methanol to remove unreacted ammonia and TEOS. Theobtained nanoparticles were vacuum-dried at room temper-ature for further use.To obtain amino-functionalized nanoparticles (SiO2@NH2),the silica nanoparticles (3.6 g) were dispersed in 120 mL ofanhydrous toluene, followed by addition of 1.2 mL of APTES.The reaction mixture was vigorously stirred for 24 h at refluxtemperature. The amino-functionalized nanoparticles werecollected by centrifugation, washed several times withmethanol and acetone, and treated as described above.Si@BiBB was prepared using the following method: 500 mgof SiO2@NH2 and 0.8 mL of triethylamine were dispersed in24 mL of THF, and the mixture was placed in an ice bath.Next, 1.15 g of BiBB was added dropwise to the suspension.The reaction mixture was warmed to ambient temperature andstirred overnight. The initiator-modified nanoparticles wereisolated by centrifugation, washed several times with methanoland water, and dried in a vacuum desiccator overnight forfurther use.2.3. Synthesis of Copolymer Brushes Grafted onSilica Nanoparticles (Si@co). Si@BiBB nanoparticles (100mg) were added to a 25 mL flask and dispersed in 4 mL ofmethanol by sonication. After addition of CuBr2 (8 mg),METAC (250 μL, 1 mmol), GMA (140 μL, 1 mmol), andPMDETA (7.4 μL) dissolved in 3 mL of water, the mixedsolution was bubbled with nitrogen for 15 min to removeoxygen. Subsequently, the reaction solution was mixed with 1mL of AscA (8 mg) to form the CuBr/CuBr2/PMDETAATRP catalyst. The reaction mixture was bubbled withnitrogen gas for another 15 min, then sealed and heated to60 °C for 24 h under a nitrogen atmosphere. The productswere isolated and purified using the same procedures asdescribed in Section 2.2.2.4. Synthesis of Copper-Doped Carbon Dots.Typically, 1.05 g of citric acid, 500 mg of Na2[Cu(EDTA)],and 5 mmol of the amine precursor ethylenediamine weredissolved in 10 mL of water in a Teflon hydrothermal synthesisreactor. The autoclave was placed in a muffle furnace andheated to 200 °C for 5 h. After the reactor was cooled toambient temperature, the suspension was centrifuged at 6000rpm for 5 min and filtered with a 0.45 μm filter membrane toremove agglomerated particles. The filtrate was transferred intoa dialysis bag (MWCO = 500 Da) and dialyzed against waterfor 24 h. CDs were obtained by freeze-drying the purifiedsuspension and then stored in a vacuum desiccator.2.5. Preparation of Polymer Brushes Modified withCDs (Si@co@CDs). Briefly, 50 mg of Si@co and 25 mg ofCDs were dispersed in 1 mL of methanol and water,respectively. Next, these two suspensions were mixed andheated to 60 °C for 24 h. The product was isolated andpurified using the same procedures as those described inSection 2.2.2.6. Preparation of Polymer Brushes Modified withCDs and Boronic Acids (Si@co@BA). To introduce boronicacid groups onto Si@co@CDs particles, Si@co@CDs (30mg), FPBA (10 mg), and NaBH3CN (5 mg) were dispersed in2 mL of ethanol. The mixed dispersion was magneticallystirred at room temperature for 24 h, and the products wereACS Omega http://pubs.acs.org/journal/acsodf Articlehttps://doi.org/10.1021/acsomega.5c01507ACS Omega 2025, 10, 14536−1454614538http://pubs.acs.org/journal/acsodf?ref=pdfhttps://doi.org/10.1021/acsomega.5c01507?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ascollected using the same procedures as described in Section2.2.2.7. Characterization. Transmission electron microscopy(TEM) characterization was carried out using a JEM-1400Plusmicroscope (JEOL, Japan). Scanning electron microscopy(SEM) was performed using a JSM-6700F instrument (JEOL,Japan). Fourier transform infrared (FT-IR) spectroscopy wasconducted with a Nicolet iS5 instrument (ThermoFisherScientific Inc., Waltham, USA). UV−vis spectroscopy wasperformed using either a Cary 60 UV−vis spectrophotometer(Agilent Technologies, USA) or a Varioska LUX multimodemicroplate reader (ThermoFisher Scientific Inc., Waltham,USA). Fluorescence spectroscopy was conducted using either aCary Eclipse fluorescence spectrophotometer (Agilent Tech-nologies, USA) or a Varioska LUX multimode microplatereader (excitation filter: 340−380 nm; dichromatic mirror: 400nm; suppression filter: 435−485 nm). Dynamic light scattering(DLS) and zeta potential measurements were carried out byusing a Zetasizer Nano ZS instrument (Malvern Instruments,UK). Nuclear magnetic resonance (NMR) spectroscopymeasurements were performed with a Bruker DRX400spectrometer at a proton frequency of 400.13 MHz. Thermalgravimetric analysis (TGA) was conducted using a TGA Q500Thermogravimetric Analyzer in an air atmosphere. Fluorescentimaging was performed using a Nikon Eclipse Ci fluorescencemicroscope (Nikon, Japan). ICP−MS analysis was conductedusing an Agilent 7850 ICP−MS.3. RESULTS AND DISCUSSION3.1. Synthesis and Characterization of Nanocompo-sites. The size and surface morphology changes at each stepduring the synthesis of multifunctional nanocomposites wereinvestigated using SEM, TEM, and DLS. As shown in Figure1a and d, the initiator-modified silica nanoparticles werespherical with a uniform diameter of around 141 nm, whichcorresponds to the hydrodynamic diameter data in Figure S1a(approximately 162 nm). After grafting METAC-GMAcopolymer brushes onto the Si@BiBB surface, the Si@conanocomposites exhibited a core−shell structure with aprominent copolymer shell (∼5 nm) visible in Figure 1e.The average diameter of Si@co was determined to be 150 nm(Figure 1b). However, the hydrodynamic diameter of Si@coappeared to be significantly larger (approximately 700 nm)than that of Si@BiBB, suggesting that the polymer brushesattached to the nanoparticles were hydrated and stretched insolution (Figure S1b). As shown in Figure S2, the morphologyof Si@co@CDs showed no obvious difference compared toSi@co under an electron microscope due to the minuscule sizeof the CDs. However, the hydrodynamic diameter of Si@co@CDs decreased by approximately 150 nm after immobilizationof the CDs, as the CDs reacted with the epoxy groups andmade the nanocomposites became more hydrophilic anduniformly dispersed. Additionally, similar morphology and sizecan be observed when Si@co@BA was inspected comparedwith Si@co (Figures 1c,f and S1c).The amount and chemical composition of the copolymer inthe nanocomposite were analyzed by using TGA and 1HNMR. As shown in Figure 2, the TGA curve slightly declinedbelow 250 °C due to the evaporation of residual organicsolvent and water during the heating process. When thetemperature increased to above 250 °C, an approximate 8.4%weight loss occurred in Si@BiBB, which can be attributed tothe surface modification by APTES and BiBB molecules. Si@co exhibited a significant weight loss of around 63.4% as thetemperature increased from 250 to 300 °C. This substantialweight loss is caused by the thermal decomposition of organiccopolymers on the core−shell nanocomposites. Consequently,the amount of copolymer in Si@co was calculated to be 52.8%based on the difference in weight loss between Si@BiBB andSi@co. In additional TGA experiments, we found that thedifference of weight loss between Si@BiBB and Si@NH2 at600 °C was 1.1%, indicating that the amount of the initiatorimmobilized on the silica nanoparticles was around 0.073mmol per gram of silica. If all the immobilized BiBB moleculesFigure 1. SEM and TEM images of (a,d) Si@BiBB, (b,e) Si@co, and (c,f) Si@co@BA. Scale bar in (a−c): 100 nm; (d−f): 50 nm.ACS Omega http://pubs.acs.org/journal/acsodf Articlehttps://doi.org/10.1021/acsomega.5c01507ACS Omega 2025, 10, 14536−1454614539https://pubs.acs.org/doi/suppl/10.1021/acsomega.5c01507/suppl_file/ao5c01507_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsomega.5c01507/suppl_file/ao5c01507_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsomega.5c01507/suppl_file/ao5c01507_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsomega.5c01507/suppl_file/ao5c01507_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig1&ref=pdfhttp://pubs.acs.org/journal/acsodf?ref=pdfhttps://doi.org/10.1021/acsomega.5c01507?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ascontributed to initiate the polymerization, the averagemolecular weight of poly(METAC-co-GMA) grafted on thesilica nanoparticles is calculated to be around 14,000 Da.Moreover, the copolymer content was further explored using1H NMR spectroscopy in DMSO-d6 (Figure S3). The methyland methylene groups from GMA exhibited two peaks at 2.07and 4.13 ppm (green and yellow dots), and the proton signalof terminal CH−CH2−O at 2.35 ppm overlapped with theDMSO-d6 peak at 2.49 ppm. For the METAC unit, the 1HNMR single peak of the methyl group and multiple peaks ofthe methylene group appeared at 1.24 and 5.77 ppm (red andblue dots), while the peak of the methyl groups from thequaternary ammonium group coincided with the water peak inDMSO-d6 at 3.3 ppm. Consequently, the final ratio of METACto GMA in poly(METAC-co-GMA) was calculated to beapproximately 1:1.6 based on the relative intensities of the 1HNMR peaks, indicating that fewer METAC monomers wereincorporated into the polymer chains during the ATRPcopolymerization.The optical properties of the copper-doped CDs wereexamined by using UV−vis absorption and fluorescencespectroscopy. Figure 3a presents the steady-state absorptionspectra of hybrid CDs. The as-prepared CDs displayed ashoulder peak in the high-energy range (240 nm) and a broadpeak at 347 nm, which can be assigned to the π−π* transitionsof sp2-hybridized carbon and n−π* transitions at the edge ofthe carbon lattice, respectively. In the wavelength region of400−550 nm, a characteristic broad and low-strengthabsorption of CDs appeared originating from low-energysub-band gaps caused by surface defects.Photoluminescence (PL) spectra of CDs are shown inFigure 3b. The optimal excitation of the CDs is located at 420nm, with an emission maximum wavelength at 470 nm, whichendows the CDs with bright blue fluorescence in aqueoussolution under 365 nm UV light. Additionally, by comparingthe PL emission peaks under different excitation wavelengthsranging from 280 to 520 nm, the excitation-dependentemission properties of the CDs, which reflect importantinformation related to different emission centers andtransitions, were further investigated. As illustrated in Figure3c, the PL peaks exhibited two separate regions: in theexcitation range of 280−400 nm, the emission positionremained at 470 nm, and PL intensities increased with therise of the excitation wavelength, revealing the dominance of asingle emissive transition. When the excitation moved above400 nm, the main emission peaks shifted toward higherwavelengths (red-shifted) along with lowered intensities.These results demonstrate the excitation-wavelength-depend-ent feature and excellent PL properties of the CDs.Based on the PL ability of CDs, the fluorescence spectra ofSi@co@CDs and Si@co@BA were measured to investigatethe CDs introduced into the nanocomposites (Figure S4a).Both Si@co@CDs and Si@co@BA exhibited strong fluo-rescence emission peaks at 470 nm under a maximumexcitation wavelength, which agreed with the pure CDs,indicating that CDs had been successfully immobilized ontothe polymer chains. ARS is commonly used to detect thepresence of boronic acid due to the formation of fluorescentboronate ester products.48 To prove that the CDs had reactedwith FPBA through the formation of a Schiff base betweenamino and aldehyde groups, the fluorescence spectra of Si@co@CDs-ARS and Si@co@BA-ARS complexes were tested bymixing the nanocomposites with ARS. As shown in Figure S4b,the color of the pinkish ARS solution was changed to orangeby the Si@co@BA nanocomposite due to formation of theboronate ester, while the ARS color turned brownish afteraddition of Si@co@CDs. Moreover, Si@co@CDs-ARSdisplayed an obvious fluorescence emission centered at 540nm due to the intrinsic fluorescence of CDs in thenanocomposites. After modification with boronic acid, theSi@co@BA particles were able to react with ARS to form aboronate ester complex, which showed a fluorescence emissionpeak red-shifted to 550 nm with an increased intensity underFigure 2. TGA analysis of Si@BiBB and Si@co.Figure 3. (a) UV−vis spectra of CDs. (b) Maximum fluorescence excitation and emission fluorescent spectra of CDs (excitation: 400 nm). (c)Excitation wavelength dependence of CDs.ACS Omega http://pubs.acs.org/journal/acsodf Articlehttps://doi.org/10.1021/acsomega.5c01507ACS Omega 2025, 10, 14536−1454614540https://pubs.acs.org/doi/suppl/10.1021/acsomega.5c01507/suppl_file/ao5c01507_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsomega.5c01507/suppl_file/ao5c01507_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsomega.5c01507/suppl_file/ao5c01507_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig3&ref=pdfhttp://pubs.acs.org/journal/acsodf?ref=pdfhttps://doi.org/10.1021/acsomega.5c01507?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as469 nm excitation. Moreover, the UV absorption of ARSbetween 400 and 500 nm, the characteristic absorption regionof the ARS-boronic acid complex, increased after Si@co@BAwas added (Figure S4c). These results confirm the successfulimmobilization of CDs and boronic acid on the nano-composites.FT-IR spectroscopy was used to explore the chemicalcomposition and functional group changes after each processstep, as shown in Figures 4a and S5. For Si@NH2, absorptionbands at 796, 949, and 1062 cm−1 corresponding to the Si−O−Si symmetrical stretching vibration, Si−OH bendingvibration, and Si−O asymmetric stretching vibration, as wellas a weak stretching vibration band of amino groups at 3348cm−1, were observed. After the acylation reaction with theinitiator, amide signals corresponding to the C�O stretchingvibration at 1635 cm−1 and N−H stretching vibration at 1534cm−1 appeared, indicating that the acylation reaction betweenBiBB and Si@NH2 occurred. After poly(METAC-co-GMA)was grafted onto the silica nanoparticles by SI-ATRP, a newband at 907 cm−1 assigned to the stretching vibration of epoxygroups from GMA and a band at 1442 cm−1 assigned to thebending vibration of the C−H bond from the quaternaryammonium groups were detected. Furthermore, a character-istic band corresponding to the stretching vibration of estercarbonyl groups at 1729 cm−1 from the two monomers alsoappeared in the IR spectra of Si@co. For the CDs, a strong N−H double peak signal around 3300 cm−1 can be seen, implyingthat the CDs possessed abundant amino groups able to reactwith the epoxy groups in the polymer brush.49 As a result, theband of epoxy groups at 907 cm−1 disappeared in Si@co@CDsafter the introduction of CDs. The similar IR results betweenSi@co@CDs and Si@co@BA may be explained by the limitedloading of boronic acid in the nanocomposite.Zeta potential was measured to further confirm thesuccessful modification during different stages, as shown inFigure 4b. Both Si@NH2 and Si@BiBB displayed a negativecharge due to the abundant Si−OH groups on the silicananoparticles. After GMA and METAC were polymerized ontothe silica core, the surface charge increased sharply to 40 mV,caused by the positively charged QACs. Due to the slightpositive charge of CDs, the Zeta potential of Si@co@CDsdecreased slightly after the surface was occupied with part ofthe CDs, which was similar to that of Si@co@BA. Finally, thecontents of copper and boron in the nanocomposites wereanalyzed by ICP−MS, which were determined to be 0.03% and0.02%, respectively, confirming the presence of the metal andboronic acid in the nanocomposites.3.2. Antibacterial Effect of Si@co@BA. Based on theantibacterial effect of the quaternary amines from the cationiccopolymer and the copper dopant in the CDs, the synergisticantibacterial capability of the nanocomposites was investigatedon E. coli using the plate counting method. As shown in FigureFigure 4. (a) FTIR spectra of Si@BiBB, Si@co, and Si@co@BA. (b) Zeta potential of Si@NH2, Si@BiBB, Si@co, Si@co@CDs, Si@co@BA, andCDs.Figure 5. (a) Photographs and (b) corresponding cell survival rate of Escherichia coli treated with different concentrations of Si@co@BA.ACS Omega http://pubs.acs.org/journal/acsodf Articlehttps://doi.org/10.1021/acsomega.5c01507ACS Omega 2025, 10, 14536−1454614541https://pubs.acs.org/doi/suppl/10.1021/acsomega.5c01507/suppl_file/ao5c01507_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsomega.5c01507/suppl_file/ao5c01507_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig5&ref=pdfhttp://pubs.acs.org/journal/acsodf?ref=pdfhttps://doi.org/10.1021/acsomega.5c01507?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as5, the number of bacterial colonies decreased gradually as theconcentration of Si@co@BA increased. When the concen-tration exceeded 1 mg/mL, Si@co@BA exhibited strongantibacterial effects against E. coli cells, as few bacterial colonieswere observed on the corresponding agar plate. Aftercalculation, the bactericidal rate was found to reach above99% when the concentration of Si@co@BA was 1 mg/mL.The strong antibacterial activity of Si@co@BA can beattributed to the synergistic effect of the high content ofMETAC and copper dopant. Therefore, 1 mg/mL was selectedas the concentration of the antibacterial material in subsequentexperiments.QACs can interact with negatively charged cell membranesthrough electrostatic interactions, leading to strong cellularlysis, while copper-doped carbon dots can kill bacteria bydestroying cell membranes and binding to bacterial proteins.50Figure 6 shows the antibacterial results of different nano-composites to demonstrate the synergistic antibacterial effect.Compared with the control group, the bactericidal rate of Si@co against E. coli was 56.4% at a concentration of 1 mg/mL,mainly due to the positive charge of the cationic copolymer.The antibacterial abilities of CDs were also explored. As shownin Figure 6a, the CDs (200 μg/mL) exhibited a highantibacterial effect with a 15.4% bacterial survival rate. Thestrong antibacterial behavior of CDs may be attributed to theirFigure 6. (a) Photographs and (b) corresponding cell survival rate of E. coli treated with 1 mg/mL of Si@co, Si@co@CDs, Si@co@BA, and 200μg/mL of CDs.Figure 7. (a−c) Photographs and (d) corresponding residual rate of remaining E. coli in the medium after treatment with Si@co@CDs and Si@co@BA. TEM images of E. coli mixed with (e) Si@co@CDs and (f) Si@co@BA. Scale bar: 1 μm.ACS Omega http://pubs.acs.org/journal/acsodf Articlehttps://doi.org/10.1021/acsomega.5c01507ACS Omega 2025, 10, 14536−1454614542https://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig7&ref=pdfhttp://pubs.acs.org/journal/acsodf?ref=pdfhttps://doi.org/10.1021/acsomega.5c01507?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-assmall size, enabling easy access to the cell membranes, andCDs could achieve 100% antibacterial efficiency at the sameconcentration (Figure S6a). When the CDs were loaded ontoSi@co, their antibacterial ability is limited but can still killbacteria by direct contact based on charge-induced physicaldestruction and reactive oxygen species-triggered oxidativestress.41 As a result, the bacterial survival rate with 1 mg/mL ofthe nanocomposite Si@co@CDs was found to be 15.2%,similar to that achieved with 200 μg/mL of CDs. This resultindicates that only a small number of CDs need to be loadedonto the nanocomposites to achieve a bactericidal effect. Forthe nanocomposite containing both CDs and boronic acids(Si@co@BA), the bactericidal rate reached 99% with 1 mg/mL of the antibacterial materials. The mechanism of bacteria-killing using the Si@co@BA nanocomposite was furtherinvestigated by measuring reactive oxygen species during thebactericidal process. As shown in Figure S6b, E. coli showedbright green fluorescence after treatment with Si@co@BA,indicating that the CDs on the nanocomposite had generatedsome reactive oxygen species for bacteria-killing. These resultssuggest that both the CDs and the boronic acids contributed toenhancing the antibacterial efficiency of the cationic copolymerin a synergistic way. In addition, Live/Dead staining wascarried out to further observe the status of E. coli aftertreatment (Figure S7a). For the bacteria treated with Si@co,half of the cells were dead, as indicated by the red fluorescence.Treatment with Si@co@CDs and Si@co@BA led to moreobvious E. coli cell death. Additionally, the morphologies of E.coli after treatment were further observed by SEM (FigureS7b). The control group showed intact bacterial structureswith smooth cell membrane surface, while partly wrinkleddeformations appeared on the cell walls after Si@co treatment.In contrast, the bacteria treated with Si@co@CDs and Si@co@BA exhibited collapsed membranes and an abnormalcellular morphology, suggesting that the integrity of thebacteria was severely damaged.3.3. Bacterial Binding Behavior of Si@co@BA. Boronicacids are known to bind to bacterial membranes by formingboronate ester bonds with the cis-diol structures on theextracellular polysaccharides on bacterial surface.51 In thiswork, Si@co@BA was designed to capture E. coli throughformation of a boronic acid−diol complex and electrostaticinteraction. To investigate the bacterial affinity of Si@co@BAfor binding and labeling bacteria, a bacterial separationexperiment was conducted. Si@co@BA was mixed with E.coli in PBS. After separation of the settlement of bacteria-Si@co@BA aggregates, the residual bacteria were quantified byplate counting. As shown in Figure 7a−d, both Si@co@CDsand Si@co@BA displayed significant bacterial trappingcapacity, while Si@co@BA exhibited a higher bacterialtrapping capacity than Si@co@CDs. This result can beattributed to the additional removal capability contributed bythe boronic acid in Si@co@BA, in addition to the electrostaticinteraction originated from the cationic copolymer.The interaction between the nanocomposites and E. coli wasfurther investigated by studying bacteria-nanocompositeaggregates using TEM. As shown in Figure 7e and f, thebacterial cells (red circles) treated with Si@co@CDs werefound to be separated from the nanocomposites and formedseveral aggregates. For bacteria treated with Si@co@BA thatcontains boronic acid, the bacilliform E. coli was closelysurrounded by many nanocomposites as the boronic acidmolecules readily facilitated the formation of the nano-composite-cell complex. Furthermore, the bacteria remainedround shaped, and no apparent cellular damage was observed,indicating that the reduction of bacteria in the supernatantmainly resulted from molecular binding rather than bacteriakilling.3.4. Labeling and Imaging of Bacteria with Si@co@BA. The microbial binding and imaging capability of thefluorescent nanocomposites were assessed using fluorescencemicroscopy of E. coli treated with the nanocomposites. Asshown in Figure 8, in the control group, the E. coli cells wereinvisible under fluorescence microscopy. After adding the twotypes of nanocomposites, the E. coli cells were effectivelylabeled with the nanocomposites and emitted intense bluefluorescence from the CDs. Flow cytometry was alsoconducted to evaluate the nanocomposites for bacterialFigure 8. Fluorescent microscope images of E. coli treated with Si@co@CDs and Si@co@BA (scale bar: 20 μm; excitation: 405 nm).ACS Omega http://pubs.acs.org/journal/acsodf Articlehttps://doi.org/10.1021/acsomega.5c01507ACS Omega 2025, 10, 14536−1454614543https://pubs.acs.org/doi/suppl/10.1021/acsomega.5c01507/suppl_file/ao5c01507_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsomega.5c01507/suppl_file/ao5c01507_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsomega.5c01507/suppl_file/ao5c01507_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsomega.5c01507/suppl_file/ao5c01507_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsomega.5c01507/suppl_file/ao5c01507_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig8&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig8&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig8&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsomega.5c01507?fig=fig8&ref=pdfhttp://pubs.acs.org/journal/acsodf?ref=pdfhttps://doi.org/10.1021/acsomega.5c01507?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asfluorescent labeling. Bacterial cells typically exhibit lowintensity autofluorescence due to the presence of endogenousfluorophores such as collagens, porphyrins, and flavins.52 Asshown in Figure S8, E. coli cells in the control group emitted aweak background fluorescence. After the addition of Si@co@CDs and Si@co@BA nanocomposites, the bacterial cellsshowed a significantly higher fluorescence. The population-intensity peaks in the flow cytometry chart of the nano-composite-treated E. coli right-shifted to form a broader peakcompared to the control sample. The result indicates a strongattachment of the nanocomposites on the surface of thebacteria membrane even under the flow condition. As a result,the fluorescent nanocomposites may be used as potentialfluorescence markers to detect living bacteria by flowcytometry based on their fluorescent imaging and bacterialbinding capacity.4. CONCLUSIONSIn conclusion, we have developed innovative multifunctionalnanocomposites capable of bacterial binding, fluorescentimaging, and synergistic antibacterial activity by combiningcationic copolymer brushes grafted onto silica nanoparticleswith copper-doped CDs and boronic acid. These hybridnanomaterials exhibit bacterial fluorescent imaging capabilitiesthanks to the incorporated CDs, enhanced affinity for bindingbacterial surfaces through boronic acid binding and electro-static interaction, and a synergistic antibacterial effect based onthe positive charge of QACs and copper-ion-inducedbactericidal action. Although the antibacterial ability of CDsis somewhat reduced after incorporation into the polymerbrushes, the nanocomposite can still kill bacteria by directcontact based on charge-induced physical destruction andreactive oxygen species-triggered oxidative stress. The multi-functional nanocomposites open new possibilities for affinityseparation, detection, and inhibition of pathogenic bacterialcells, shedding new light on the development of innovativeantibacterial materials and other biological research platformssuch as glycan labeling and synergistic treatment of cancercells.■ ASSOCIATED CONTENT*sı Supporting InformationThe Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acsomega.5c01507.Details and additional results of binding analysis,boronic acid determination, bacteria cultivation, anti-bacterial activity assay, and characterization of Si@co@BA (PDF)■ AUTHOR INFORMATIONCorresponding AuthorLei Ye − Division of Pure and Applied Biochemistry,Department of Chemistry, Lund University, Lund 22100,Sweden; orcid.org/0000-0002-3646-4072;Email: lei.ye@tbiokem.lth.seAuthorsQicheng Zhang − Division of Pure and Applied Biochemistry,Department of Chemistry, Lund University, Lund 22100,SwedenSi Chen − Polymer & Materials Chemistry, Department ofChemistry, Lund University, Lund 221 00, SwedenXiaoting Xue − Polymer & Materials Chemistry, Departmentof Chemistry, Lund University, Lund 221 00, SwedenSolmaz Hajizadeh − Division of Pure and AppliedBiochemistry, Department of Chemistry, Lund University,Lund 22100, Sweden; orcid.org/0000-0002-0348-8756Tomohiko Yamazaki − Research Center for Macromoleculesand Biomaterials, National Institute for Materials Science(NIMS), Tsukuba 305-0047, Japan; orcid.org/0000-0003-2136-8042Complete contact information is available at:https://pubs.acs.org/10.1021/acsomega.5c01507NotesThe authors declare no competing financial interest.■ ACKNOWLEDGMENTSThe authors are grateful for financial support from the SwedishResearch Council VR (grant number 2019-04228) and theRoyal Physiographic Society in Lund. 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