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Gabriel Tai Huynh, Jun Qiu, Edith van den Bosch, [Tomohiko Yamazaki](https://orcid.org/0000-0003-2136-8042), [Chiaki Yoshikawa](https://orcid.org/0000-0002-6589-387X)

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[Concentrated Polymer Brush-Modified Magnetic Particles for a Diagnostic Immunoassay](https://mdr.nims.go.jp/datasets/47902b69-0556-4f08-85c8-0f052cc1278f)

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Concentrated Polymer Brush-Modified Magnetic Particles for a Diagnostic ImmunoassayConcentrated Polymer Brush-Modified Magnetic Particles for aDiagnostic ImmunoassayGabriel Tai Huynh, Jun Qiu, Edith van den Bosch, Tomohiko Yamazaki, and Chiaki Yoshikawa*Cite This: Langmuir 2025, 41, 32432−32442 Read OnlineACCESS Metrics & More Article Recommendations *sı Supporting InformationABSTRACT: Nonspecific protein absorption is an ongoingproblem in the development of highly sensitive magnetic particle(MP)-based diagnostic assays, whereby the MP surface undergoesbiofouling, significantly reducing the limit of detection andsensitivity of the device. In this study, a bioinert, concentratedpolymer brush (CPB) composed of poly[poly(ethylene oxide)methyl ether methacrylate] (PPEGMA) was employed to reducethe absorption of protein onto the MP surfaces; and an anti-humanserum albumin antibody (Ab) was then immobilized onto thebrush layer by click chemistry to demonstrate its application as animmunoassay platform. The amount of antibody grafted onto theends of the brush coating was quantified by a BCA assay with ahigh grafting efficiency (∼0.80 antibody per chain). Furthermore,when using the antibody-conjugated CPB-coated MPs (MP-PPEGMA-Ab) as an immunoassay platform, we were able to determinethe capture efficacy of human serum albumin (HSA) in both a buffered solution and diluted human serum by colorimetric analysis,and further confirmation was achieved via liquid chromatography/mass spectrometry, in which our MPs showed a high selectivitytoward the targeted analyte. Lastly, we demonstrated that MP-PPEGMA-Ab was capable of detecting human serum albumin at aconcentration as low as 6.4 ng mL−1, 30% more sensitive than the unmodified MP, demonstrating the impact of CPBs on diagnosticassays. Due to their high selectivity and sensitivity, our CPB-based MPs are expected to be applicable for a wide range ofimmunoassay applications by employing different bioinert polymers and biofunctional groups.1. INTRODUCTIONMagnetic particles (MPs) have been used extensively inbiomedical applications, as either a contrast agent for deeptissue imaging,1−3 cancer therapeutic (hyperthermia)agents,4−6 or drug carriers for cancer treatment or forselectively capturing and/or separating cells,7 proteins, and/or DNA from complex biological matrices. Compared to otherpurification techniques such as ionic precipitation, dialysis, andelectrophoresis,8−10 MP separation requires minimal technicalexpertise, can be easily scaled, and does not require a largecapital investment. Therefore, MPs have been used in multipleapplications for selective capture and separation, such ascapturing and separating tumor cells from blood,11,12 extractingantigens for viral detection,11,13,14 or concentrating low-abundance proteins in developing highly sensitive assays.15These surfaces are typically modified with bioactive groupssuch as antibodies or peptides, which selectively bind to thetarget molecule, allowing for easy capture and subsequentextraction. Conversely, due to the complex nature of biologicalsolutions, such as protein-rich serum, nonspecific proteinadsorption and interactions would inevitably lead to poorbinding and poor sensitivity and selectivity. Therefore, in orderto enhance the selectivity and sensitivity of biosensors, bioinertcoatings have been explored to prevent and minimizenonspecific protein absorption.Polymer coatings of biocompatible hydrophilic polymerssuch poly(ethylene) glycol (PEG) or zwitterionic poly-mers16−18 have been used to suppress nonspecific proteinadsorption. Typically, these polymers are grafted onto thesurface through physisorption19,20 or chemical conjugation ofpreformed polymer chains.21 However, the efficacy of thesecoatings can greatly vary for several reasons, such as thepolymer chain length,22,23 grafting density24,25 and spacing,26,27polymer orientation and conformation,28,29 and chemicalstability30 all affecting their performance. Recently, surface-initiated atomic transfer radial polymerization (SI-ATRP) hasbeen explored as a method for grafting polymers, such asPEG31,32 or zwitterionic33−36 polymers onto surfaces,35producing well-defined polymer brush structures with a highReceived: August 12, 2025Revised: October 24, 2025Accepted: November 11, 2025Published: November 25, 2025Articlepubs.acs.org/Langmuir© 2025 American Chemical Society32432https://doi.org/10.1021/acs.langmuir.5c04175Langmuir 2025, 41, 32432−32442This article is licensed under CC-BY 4.0Downloaded via NATL INST FOR MATLS SCIENCE (NIMS) on December 12, 2025 at 06:53:07 (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="Gabriel+Tai+Huynh"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Jun+Qiu"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Edith+van+den+Bosch"&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="Chiaki+Yoshikawa"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/showCitFormats?doi=10.1021/acs.langmuir.5c04175&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?goto=articleMetrics&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?goto=recommendations&?ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?goto=supporting-info&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=tgr1&ref=pdfhttps://pubs.acs.org/toc/langd5/41/48?ref=pdfhttps://pubs.acs.org/toc/langd5/41/48?ref=pdfhttps://pubs.acs.org/toc/langd5/41/48?ref=pdfhttps://pubs.acs.org/toc/langd5/41/48?ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c04175?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://pubs.acs.org/Langmuir?ref=pdfhttps://pubs.acs.org/Langmuir?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/grafting density and good long-term stability.37 Unlikeconventional radial polymerization, the surface is modifiedwith an alkyl halide group, whereby the distribution of thedecapping and capping of the halogen initiator can becontrolled, which allows for high-density polymer grafting.SI-ATRP has been successfully applied to silicon38 and goldsurfaces,39,40 silica nanoparticles,41,42 and graphene oxide,43where these surfaces were modified to minimize proteinadsorption. The Tsujii group has extensively explored the useof high-density polymer brushes, otherwise known asconcentrated polymer brushes (CPBs),44 for antifoulingapplications. They have reported that CPBs form uniquehigh-extension structures when swollen in aqueous solvents,allowing them to have strong durability, long-term stability,and a super lubrication effect, giving rise to their antifoulingproperties. Moreover, we recently demonstrated the uniquesize-exclusion effect of CPBs by varying both the graft densityand chemical composition of various polymers, whileconfirming that CPBs significantly suppressed proteinadsorption and subsequent cell adhesions when directlycompared against thin films and coatings with a semidilutepolymer brush (SDPB) configuration of the same correspond-ing polymers.38,42,45 Such bioinert CPBs are expected to beuseful base coating to enhance selective binding of thefunctional groups on MPs (Scheme 1).In this work, we developed CPB-modified MPs that canselectively capture and concentrate a target protein. WhileCPB-modified magnetic particles have been seen countlesstimes in the literature,46−48 its use as an antifouling coating forimproving immunoassays, to the best of our knowledge, has yetto be realized. By using commercially available hydroxyl-functionalized organosilica-coated MPs, we first graftedpoly[poly(ethylene glycol) methyl ether methacrylate] (PPEG-MA), which has been reported as one of the biocompatiblepolymers, onto the surface using SI-ATRP, which wepreviously reported to have good stability and functionallifetimes when previously grafted onto silica particles,37,49 orwhen used as a preventative coating within in vivo settings.42Subsequently, an antibody for targeted antigen capture wasimmobilized at the chain end of grafted PPEGMA by clickchemistry. Here, the targeted analyte was chosen to be humanserum albumin (HSA) as a representative target due to its highabundance in serum. Finally, we confirmed that the presence ofCPB on MPs improved the sensitivity for immunoassays byindirect and direct analysis (Scheme 2). Following incubationof the MPs with human serum and plasma, (1) unreacted HSAin serum and plasma was quantified with an enzyme-linkedimmunosorbent assay (ELISA) (indirect analysis) and (2)HSA bound on the MPs was quantified by direct enzyme−substrate binding, namely, adding enzyme and substrate to thesolution of MPs (direct analysis). The direct analysis showedthat MPs with CPB had a lower limit of detection of 6.4 ngmL−1, approximately 30% more sensitive than MPs without theCPB coating.As a proof of concept, in this work, we used PPEGMA as abioinert polymer and HSA as a target antigen. Since our MPcoated with a CPB layer offers a wide range of designpossibilities, by varying the type of polymer and biofunctionalgroups, this work provides a stepping stone for developing newand highly sensitive diagnostic tools for various diseases andillustrates the importance of bioinert coatings for immuno-assays.2. MATERIALS AND METHODS2.1. Materials. Ethyl-2-bromoisobutyrate (EBIB, 98.0%, TokyoChemical Industry Co., Ltd. (TCI), Japan), copper(I) bromide(Cu(I)Br, 99.9%, Wako Pure Chemical Industries, Ltd., Japan), 2,2′-bipyridyl (Bpy, 99.5%, Nacalai tesque, Japan), and N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 99.0%, Tokyo ChemicalIndustry Co., Ltd. (TCI), Japan) were used as received. {[(2-Bromo-2-methylpropionyl)oxy]propyl} triethoxysilane (BPE) was synthe-sized according to the literature.41 Poly(ethylene oxide) methyl ethermethacrylate (average Mn of ∼475) (PEGMA Sigma-Aldrich, Osaka,Japan) was purified by being passed through neutral alumina.Hydroxyl-functionalized organosilica-coated iron oxide magneticparticles (MPs) (SiMAG-Hydroxyl, diameter of 500 nm) werepurchased from chemicell GmbH (Berlin, Germany).2.2. Immobilization of BPE on MPs. SiMAG-hydroxyl particles(MPs) were washed with absolute ethanol (EtOH) three times. Thenthe MPs (100 mg), aqueous ammonia (28% (w/w) in water, 1.52 g),and EtOH (11.00 mL) were placed in a flask. BPE (0.22 g) in EtOH(2 mL) was then added to the flask, and its contents were mixed atroom temperature for 18 h. After the reaction, BPE-modified MPswere washed with absolute ethanol three times. Subsequently, theMPs were washed with methanol (MeOH) twice, and theconcentration of MPs in MeOH was adjusted to 5.0% (w/v). TheBPE-modified MPs (MP-Br) in MeOH were stored at 4 °C until use.Scheme 1. Graphical Representation of the Scope of theStudyaaOrganosilica-modified iron oxide (Fe3O4)-based magnetic particles(MPs) were modified with a concentrated polymer brush (CPB)coating to prevent nonspecific protein adsorption and subsequentlymodified with an antibody for selective antigen capture.Scheme 2. Selective Capture and Subsequent Analysis ofMP-PPEGMA-Ab toward the Targeted AnalyteaaHere, MP-PPEGMA-Ab is incubated in a protein solution, wheremagnetic separation is applied, separating both captured andnoncaptured proteins in solution.Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c04175Langmuir 2025, 41, 32432−3244232433https://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=sch1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=sch1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=sch2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=sch2&ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c04175?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as2.3. SI-ATRP of PPEGMA onto MPs. Concentrated PPEGMAbrushes were grafted onto MPs by surface-initiated atom transferradial polymerization (SI-ATRP). Briefly, MP-Br (25 mg) wasdispersed in a N2-purged MeOH solution (6.60 g) of PEGMA(6.60 g, 13.94 mmol), Cu(I)Br (10.0 mg, 0.070 mmol), Bpy (21.78mg, 0.139 mmol), and free initiator EBIB (13.59 mg, 0.070 mmol),and the solution was stirred at 30 °C for 3.5 h. After polymerization,the modified particles were washed in methanol, and theconcentration of MP-PPEGMA-Br was adjusted to 10 mg mL−1 inMeOH and stored at 4 °C until use. After polymerization, thenumber-averaged molecular weight (Mn) and the weight-averagedmolecular weight (Mw) of free polymers were determined by gelpermeation chromatography (GPC) using N,N-dimethylformamide(DMF) with 10 mM lithium chloride (LiCl), with poly(methylmethacrylate) (PMMA) calibration standards. Free polymer con-version was determined by proton nuclear magnetic resonance (1HNMR) measurements in deuterated chloroform. The theoretical Mn(Mn,conv) was calculated by= [ ] [ ] × ×M CM / EBIB MWn,conv 0 0 (1)where MW is the molecular weight of the PEGMA monomer and C isthe monomer conversion (per 100%) determined by 1H NMR.The amount of grafted PPEGMA (weight loss) was estimated bythermal degradation−differential thermal analysis (TG-DTA,TG8120, RIGAKU Co., Ltd., Tokyo, Japan). Grafting amount σ(number of chains per square nanometer) was estimated by= AN M S/( )A n,conv (2)where A is the graft amount (grams per gram) and S is the particlesurface area (square nanometers per gram).The dimensionless graft density (σ*) was calculated by* = a2 (3)where a2 is the cross-sectional area per monomer.2.4. Terminal Azidation of PPEGMA Brushes. MP-PPEGMA-Br (20 mg) particles were suspended in 1.00 g of DMF before beingmixed with a solution of DMF (1.00 g) containing sodium azide(NaN3, 0.267 mg, 4.1 × 10−3 mol). The reaction mixture was thenmixed at 50 °C for 18 h. After the reaction, MP-PPEGMA-N3 wasthoroughly washed with DMF before being adjusted to aconcentration of 10 mg of MP-PPEGMA-N3 in 1 g of DMF andstored at 4 °C until use.2.5. Azide−Alkyne Click Chemistry for PPEGMA Function-alization. N-(4-Pentynoyloxy) succinimide (30.7 mg), Cu(I)Br (75mg), PMDETA (90.6 mg), and DMF (7.50 g) were placed in aFigure 1. Synthesis of MP-PEGMA-Ab, where we surface modified commercially available hydroxyl-functionalized organosilica magneticmicroparticles with (I) a bromide initiator, (II) an antifouling coating, (III) an azide terminating group, (IV) a NHS terminating group, and (V) anantibody. aAzidation conversion was confirmed via 1H NMR (Figure S3b), and bazide−alkyne click chemistry conversion was confirmed via 1HNMR (Figure S3c). The structure of SiMAG-OH was provided by the commercial supplier.Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c04175Langmuir 2025, 41, 32432−3244232434https://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=fig1&ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c04175?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asSchlenk tube, and then, the MP-PEGMA-N3 in DMF (75 mg in 75 gof DMF) was added to the tube. The mixture was vigorously mixed atroom temperature for 18 h. After the click reaction, the resulting MP-PPEGMA-NHS was washed with MeOH. The MP-PPEGMA-NHSwas then dispersed in dimethyl sulfoxide (DMSO) (10 mg of MP-PEGMA-NHS in 1 g of DMSO) and kept at 4 °C until use.2.6. Fixation and Quantification of the Antibody on MPs.MP-PPEGMA-NHS and pristine MPs were first suspended in 25 mMMES-NaOH buffer (10 mg mL−1), before the addition of 400 μL ofthe goat anti-human albumin antibody (HSA antibody, 1 mg mL−1,Bethyl Laboratories, Inc., Montgomery, TX). The solution was thenmixed at 4 °C for 1 h. Following the reaction, the samples werecentrifuged at 15 000 rpm for 5 min, where the amount of unboundantibodies present within the supernatant was quantified through amicrobicinchoninic acid (micro-BCA) assay (Thermo FisherScientific, Waltham, MA). Subsequently, the MP-antibody particleswere washed thoroughly with fresh HEPES buffer (10 mM HEPES-NaOH (pH 7.9)), 50 mM potassium chloride (KCl), 1 mMethylenediaminetetraacetic acid (EDTA), and 10% (v/v) glycerin.The modified particles were adjusted to a concentration of 10 mgmL−1 and stored at 4 °C.2.7. Human Serum and Plasma Protein Adsorption Testing.MP-PPEGMA-Ab particles (5.0 mg) was first suspended in 100 μL ofa 1× phosphate-buffered saline (PBS) solution. Then, 900 μL ofhuman reference serum (RS10-110-4, Bethyl Laboratories, Inc.) orhuman whole plasma (human EDTA-2Na plasma, single donor)(KAC Co., Ltd., Kyoto, Japan) was added to the MP solution. Themixture was then incubated at 37 °C for 1 h, before being washed fivetimes with 1 mL of 1× PBS to remove all unbound proteins. Afterbeing washed, the MPs were centrifuged at 15 000 rpm for 4 min, andthe supernatant was discarded. The proteins bound to the surfacewere quantified by first adding 500 μL of a 5.0% (w/v) sodiumdodecyl sulfate (SDS) solution to the captured magnetic particles.The solution was mixed for 1 h, allowing all of the bound proteins todetach from the surface. Then, the supernatant was collected bycentrifugation, where the amount of human albumin was quantified byan ELISA (E80-129, Human Albumin ELISA Quantitation Set, BethylLaboratories, Inc.).2.8. Gel Electrophoresis. Following adsorption of the protein tothe magnetic particles, the concentration of the particle suspensionwas adjusted with 1× PBS to a final concentration of ∼0.25 mg μL−1.In a typical run, 10 μL of the solution was loaded into apolyacrylamide gel (NuPAGE 4−12% Bis-Tris gel, Thermo Fisher).Gel electrophoresis was conducted in accordance with themanufacturer’s provided protocol. Afterward, the bands werevisualized by silver staining (AE-1360 EzStain Silver, ATTO, Japan)and imaged with a scanner (CanonScan8800F, Canon, Japan).2.9. Direct Measurement of Albumin Captured on the MPs.Prior to the experiment, the amount of albumin presented in humanwhole plasma (human EDTA-2Na plasma, single donor) (KAC Co.,Ltd.) was determined to be 65 mg mL−1 by an ELISA. Following this,human whole plasma was diluted in the sample diluent solution (50mM Tris-HCl, 0.14 M sodium chloride (NaCl), 1.0% (w/v) bovineserum albumin (BSA), and 0.025% (v/v) Tween 20 (pH 8.0), BethylLaboratories, Inc.), as described by the manufacturer’s protocol. Thesample was diluted until the final concentration of human albuminpresent was 1.00 mg mL−1 (1000 000 ng mL−1).Then, 5.0 mg of either MP-PPEGMA-Ab or pristine MP-Ab wassuspended in 100 μL of 1× PBS, before the addition of 900 μL of thediluted human whole plasma solution. The solution was mixed andincubated at 37 °C for 1 h. Afterward, the MPs was washed five timeswith 1 mL of 1× PBS to remove any unbound proteins, before beingresuspended in 1.0 mL of 1× PBS.Then, 500 μL of the resuspended particle solution was mixed with50 μL of a solution containing a horseradish peroxide (HRP)-conjugated anti-HSA detection Ab solution (Bethyl Laboratories,Inc.). The solution was mixed for 1 h at room temperature, beforebeing centrifuged and washed 10 times in 1.0 mL of an ELISAwashing buffer solution (Bethyl Laboratories, Inc.). Subsequently, 50μL of a 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution wasadded to the particle suspension and mixed for 10 min, before 25 μLof a stop solution (Bethyl Laboratories, Inc.) was added to the MPsuspension. The amount of HSA present on the surface of themagnetic particles was then quantified by the absorbance at 450 nm,where 75 μL of the particle solution was added to a 96-well plate.3. RESULTS AND DISCUSSION3.1. Synthesis and Characterization of CPB-ModifiedMPs. The surface modifications of the hydroxyl-functionalizedorganosilica-coated MP are shown in Figure 1. Organosilica-modified magnetic particles were selected due to the ease ofthe subsequent surface modification and good colloidalstability.50 In order to first graft our polymers onto theorganosilica-MP surface, we began by modifying the surfacewith BPE, a fixed initiator for ATRP (Figure 1, I). Next,PEGMA was grafted onto the surface through SI-ATRP(Figure 1, II). The characterization of the PPEGMA coatingon the magnetic particles can be found in Table S2. Here, thenumber-weighted average molecular weight (Mn) andpolydispersity (Mw/Mn) were determined from the freepolymer formed during the reaction. It has been widelyestablished that both the Mn and the Mw/Mn of the freepolymer are highly representative of both the Mn and the Mw/Mn of the grafted polymers formed via SI-ATRP,51,52 especiallythen compared and validated against high-resolution surfacecharacterization techniques, such as X-ray photoelectronspectroscopy,53 atomic force microscopy,41,54 and transmissionelectron microscopy.41,55 Subsequently, we calculated thegrafting density (σ) and its dimensionless counterpart (σ*)to confirm that the coating on our particles consisted of CPBs(σ* > 0.1) (Table S2).A schematic representation of the classification of the brush-like structure is shown in Scheme 3, where the distancebetween the anchor site (d) and the distance between theelongated polymer chains (D) are determining factors withrespect to whether a polymeric coating is classified as CPB orSDPB.51 If the distance between two attachment points is inparity with the distance between neighboring polymer chains(D ≈ d), a repulsion barrier effect occurs in which proteins areunable to directly adsorb to the surface. Conversely, if thedistance between the polymer chains is larger than the distancebetween neighboring anchor sites (D > d), proteins can diffuseScheme 3. Schematic Representation of the Differencebetween Concentrated Polymer Brush (CPB) Structuresand Semidilute Polymer Brush (SDPB) Structures When ina Well-Dispersed SolventLangmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c04175Langmuir 2025, 41, 32432−3244232435https://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=sch3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=sch3&ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c04175?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asthrough the brush-like structure, with smaller proteins beingable to foul the surface.Previously, we reported that CPBs grafted on both siliconwafers and silica particles exhibit excellent low biofoulingcapacities when compared against their corresponding SDPBcounterparts,42 suggesting that our coatings would exhibitsimilar behavior. To validate our hypothesis, we comparedboth SDPB- and CPB-modified particles by incubating them inundiluted fetal bovine serum (FBS) for one hour beforeperforming gel electrophoresis. As shown in Figure S1,particles modified with a SDPB coating (σ = 0.009 chainnm−2, and σ* = 0.03) showed evidence of biofouling (lane 3),whereby particles with a CPB coating (σ = 0.05 chain nm−2,and σ* = 0.16 (lane 2); σ = 0.58 chain nm−2, and σ* = 1.0(lane 4)) showed no evidence of biofouling, conferring theantifouling effect from CPB coatings. It should be noted thatwhile there was batch-to-batch variation between each particleset (Tables S2 and S3), mainly differences in the number-weighted average molecular weight values. However, as thedefinition of CPB and SDPB is dependent on thedimensionless grafting density value (σ*), the observeddifferences between Mn values did not affect the overallperformance of the coating (Figure S1).Next, to functionalize the particle surface with an antibody,we modified the terminal bromide group along the PPEGMAbrushes with sodium azide, where the nucleophilic substitution(SN2) reaction resulted in the polymer brush containing anazide terminal moiety56 (Figure 1, III). The azide group wasconfirmed through FTIR analysis, with the latter showing aslight absorption peak around ∼2100 cm−1 (Figure S2).Furthermore, 1H NMR analysis on the free polymer chainfurther confirmed that the substitution reaction was approx-imately 100% (Figure S3). Previous studies by Sakakibara et al.similarly reported that the bromine at the chain end ofconcentrated PPEGMA brushes grafted on a silicon wafercould be converted into an azide group with approximately100% efficiency, further validating our observations.57 There-fore, we considered that the azidation reaction would alsoproceed on our grafted polymers on the MPs.Then, azide−alkyne click chemistry58 between N-(4-pentynoyloxy) succinimide was used to further modify thePPEGMA brush to have a terminal hydroxy-succinimidegroup, which readily reacts with primary amines on thecaptured antibody (Figure 1, IV). 1H NMR analysis of the freepolymer revealed that most terminal azides reacted with alkynehydroxy succinimide (Figure S3). Finally, we immobilized ananti-human albumin antibody (Ab) on the brush surface usingthe N-hydroxysuccinimide (NHS) group at the chain end(Figure 1, V). The amount of Ab on the MP surface wasquantified by the BCA assay. The molar ratio of the Ab tografted polymer chains was estimated to be between 0.73 and0.86, signifying that a majority of the chains had beensuccessfully modified (Table S4).3.2. Identification of Adsorbed Proteins on MP-PPEGMA-Ab. To show the capabilities of our particles forspecific analyte targeting, we used human serum albumin as amodel target. HSA is one of the most abundant proteinspresent in serum, but low levels of albumin (hypoalbumine-mia) have been associated with different diseases such ascardiovascular disease59,60 or kidney/renal disease.61 Tocapture HSA from human serum, we used an anti-HSAantibody (Ab) and subsequently attached it to our magneticparticles. Sodium dodecyl sulfate−polyacrylamide gel electro-phoresis (SDS−PAGE) was performed to confirm the captureof the protein, where proteins are separated based on theirrelative size under an applied voltage (Figure S4). As HSA hasa well-known size (67 kDa), we could easily identify thepresence of the protein by gel electrophoresis. Forexperimental controls, we ran both anti-HSA Ab at aconcentration of 1.0 mg mL−1 and MP-PPEGMA-Ab (FigureS4, lanes 2 and 3) and found that there was a noticeable bandvisible at ∼55 kDa, likely due to the light chains from the Abcomplex. Similarly, by running human reference serum (FigureS4, lane 5), we saw multiple bands, which indicated thecomplex protein matrix of human serum. Subsequent geldigestion and LC-MS on MP-PPEGMA-Ab confirmed thepresence of Ig λ chain V-III (Table 1, region 5), furthervaliding the presence of anti-HSA on the surface of theparticles. Interestingly, the top three scoring proteins taggedfrom the MP-PPEGMA-Ab (Table 1, region 5) were allassociated with actin and cardiac cells62−64 (Shroom3, actin,aortic smooth muscle, and actin, α cardiac muscle), which canbe found in human blood and serum.65 As the antibodysolution (Table 1, regions 3 and 4) showed the presence ofserum, it is likely that the appearance of these actin-relatedproteins would have been associated with it. Moreover,hemoglobin, fetal subunit β, and α-1-antiproteinase werepresent in the top scoring proteins within the protein solution.With these proteins being associated with blood,66 serum, andthe immune response,67,68 the presence of these proteins wasexpected. Similarly, as actin plays a key role in the adhesion ofcell to surfaces,69 it is unsurprising that these proteins would bepresent, likely competing against terminal NHS polymerchains.Similarly, in regions in which MP-PPEGMA-Ab was used tocapture HSA (Table 1, regions 17, 21, 25, and 28), we alsoobserved the presence of protein shroom, which is alsoassociated with the actin protein. We believe the presence ofthis protein was linked with the antibody conjugate.Furthermore, MP-PPEGMA-Ab was also incubated indifferent concentrations of human reference serum, followedby washing steps to remove any unbound proteins, and thenloaded into the gel. Here, two distinct bands were observedTable 1. Top Three Scoring Proteins Found following GelDigestion and LC-MSagel regiontop scoringprotein second top scoring proteinthird topscoringprotein3 serum albu-minα-1-antiproteinase hemoglobinfetalsubunit β4 hemoglobinfetalsubunit βserum albumin −5b shroom3 actin, aortic smooth muscle actin, α car-diac muscle11, 16, 20,24, and27serum albu-min− −12 serum albu-mintransient receptor potential cati-on channel subfamily Vmember 5proteinshroom17, 21, 25,and 28proteinshroomserum albuminaThe corresponding gel can be found in Figure S5. bThe Ig λ chain V-III region LOI was present, identified as the 12th top scoring protein.Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c04175Langmuir 2025, 41, 32432−3244232436https://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c04175?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as(Figure S5, lanes 6−10), which correspond to both HSA(Table 1, regions 16, 20, 24, and 27) and Ab (Table 1, regions17, 21, 25, and 28). As each lane was loaded with MP-PPEGMA-Ab, which had been incubated with humanreference serum and subsequently washed, we concluded thatthe HSA present was conjugated with MP-PPEGMA-Ab, withthe antibody fragments appearing as the secondary band at 55kDa. Since no protein bands except for HSA and Ab wereobserved in the eluate from MP-PPEGMA-Ab and most of theproteins identified by LC-MS analysis were HSA, weconcluded that nonspecific adsorption is efficiently suppressedby the CPB layer.Additionally, no cross-reactivity was observed with our MP-PPEGMA-Ab against other albumins (Figure S6). Fetal bovineserum (FBS) was selected as it was a good candidate, mainlydue to the presence of bovine serum albumin (BSA), whichhas a molecular size similar to that of HSA (67 kDa), whilecontaining other proteins that may nonspecifically adsorb tothe surface of the particles. As expected, we did not observeany cross-reactivity of our particles in the FBS solution, as wemodified our particles with antibodies to specifically captureHSA (Figure S6, lane 4). Additionally, we found that there wasno evidence of nonspecific protein fouling on the particlesurface (Figure S7). Interestingly, when we incubated ourmagnetic particles in 0.1% (v/v) HSA in FBS, we foundevidence of nonspecific protein absorption to our particlesmodified with BPE (Figure S7, lane 1). However, once wemodified the surface with PPEGMA, we saw a large reductionin protein adsorption, due to the hydrophilic nature ofPPEGMA, where the presence of the polymer brush structureminimized nonspecific adsorption. Moreover, following themodification of these polymer brushes with an anti-HSAantibody, we saw the presence of our proteins via SDS−PAGE,where there was a visible band present (Figures S7 and S8).3.3. Indirect and Direct Quantification of HumanSerum Albumin on MP-PPEGMA-Ab. To confirm thecapture efficiency of our MP-PPEGMA-Ab system toward thetargeted analyte, we performed an ELISA on both the solutionsupernatant (indirect) and our particle scaffold (direct)(Scheme 2; the red dotted circle indicates indirect analysis,and the green one direct analysis). First, we measured theprotein content of the supernatant (indirect), where wedetermined the amount of protein captured on the surface ofour particles by a simple mass balance (Figure 2A). It has beenpreviously reported that the presence of nanoparticles insolutions can cause light scattering artifacts that can affect theabsorption measurement.70 By incubating our particles withhuman serum containing known concentrations of HSA, wecan separate captured HSA from the solution phase by using asimple magnetic force. Subsequently, this allowsus to quantifyany remaining HSA present within the serum solution, wherewe indirectly quantify the amount of HSA captured by ourparticle system. After adding our antibody-modified CPBmagnetic particles to human serum with varying concen-trations of HSA, ranging from 4 to 86 ng mL−1 for one hour,subsequent ELISAs revealed that all residual supernatantsolutions contained concentrations of HSA that were lowerthan the working detection limit of the assay (<3.1 ng mL−1).In other words, more than 96% of the HSA present in theserum was bound to MP-PPEGMA-Ab. As shown in Figure 2a,we saw a linear relationship between the concentration of HSAinitially in solution and HSA captured on the surface, wherethe capture efficiency was ∼100%. While we only testedsamples containing less than 100 ng mL−1, it is possible thatthe dynamic range of our platform is greater than 100 ng mL−1as the surface may not be fully saturated with HSA, where thetheoretical saturation limit of our modified particles wasdetermined to be approximately 1300 μg mL−1, as shown inTable S4. This is likely due to the orientation and structure ofthe PPEGMA CPB structure, resulting in a high concentrationof Ab located on the surface. However, it should be noted thatthe actual saturation limit would be lower than the theoreticalvalue as the orientation or structure of the antibody maychange during the conjugation step, and further testing wouldbe needed to confirm the true dynamic range of the sensor.Because of the high capture efficiency of our particles, wethen used our MP-PPEGMA-Ab as a platform for a sandwichELISA (direct). Unlike conventional sandwich ELISAs, whichFigure 2. Human serum albumin capture efficiency using MP-PPEGMA-Ab in human serum, measured by (a) an indirect ELISA or(b) a direct ELISA. (a) ELISA on the supernatant followingincubation with MP-PPEGMA-Ab and (b) sandwich ELISA usingMP-PPEGMA-Ab as the substrate. The best fit line was determined tobe absorbance = 0.012[HSA] + 0.71.Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c04175Langmuir 2025, 41, 32432−3244232437https://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=fig2&ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c04175?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asinvolve multiple laborious steps, such as surface modificationand blocking, these particles can be used directly as the ELISAplatform, thus simplifying the process (Figure S9).By incubating our MP-PPEGMA-Ab with different concen-trations of human reference serum containing knownconcentrations of HSA and applying an external magneticforce, we could concentrate HSA while separating all unboundproteins in the serum. Then by incubating these particles withan HRP-conjugated antibody and performing a sandwichELISA, we could directly measure the antigen concentrationusing TMB. As shown in Figure 2b, we would detect varyingantigen concentrations, where we then calculated the limit ofdetection (LOD). As the limit of detection was defined as themean value of the background measurements plus three timesthe standard deviation, resulting in an LOD of 10 ng mL−1.3.4. Indirect and Direct Quantification of HSA inWhole Human Plasma on MP-PPEGMA-Ab. As the CPBssynthesized onto our MPs showed excellent antifoulingbehavior toward nonspecific adsorption, we tested ourdiagnostic assay in human plasma. Human plasma is a complexbiological fluid that contains a multitude of differentcomponents, such as proteins, such as fibrinogen and albumin,and immunoglobulins.71 Because of this, being able to detectkey analytes in serum has been reported to be difficult withoutsample purification, where nonspecific absorption has beenreported to affect the sensitivity and functionality of biosensorsin the past.72−74We first confirmed whether the MP-PPEGMA-Ab systemcould be used to capture HSA in whole human plasma by anindirect ELISA. Here, we incubated our particles in differentconcentrations of human whole plasma, whereby theconcentration of HSA was quantified prior to use (65 mgmL−1, as described in the Materials and Methods). Byincubating our particles for one hour, we measured thesupernatant solution to determine the amount of uncapturedHSA present in the remaining supernatant solution. As shownin Figure 3a, we found that our MP-PPEGMA-Ab couldreliably capture HSA in unpurified plasma, where there was nodifference in the trends between HSA in a single solution, orwithin the plasma/complex system. This suggests that thepresence of the concentrated polymer brush structureminimized nonspecific protein absorption, allowing for ahigher analyte sensitivity. We then measured the concentrationof HSA on the surface of our MP-PEGMA-Ab system with amodified sandwich ELISA (Figure 3b; see Figure S9 for moredetails). Here, by using the magnetic particles as the platformfor the assay, we incubated the particles with an HRP-modifiedantibody, whereby the concentration of HSA on the surfacewas determined using TMB. Here we found that theabsorbance linearly decreased with decreasing plasma concen-tration. This indicates that MP-PEGMA-Ab could accuratelydetermine the concentration of HSA in whole human plasma,where the presence of the CPB structure would minimizenonspecific protein interactions on the surface, allowing for ahigher sensor sensitivity. Subsequent calculations revealed thatthe LOD for HSA in whole human plasma was 6.4 ng mL−1,which is comparable to or better than those from recent HSAdetection studies, where concentrations in the milligram permilliliter range have been cited.75−77 To further support thisobservation, we used bare organosilica-modified MPs withoutthe PPEGMA brushes (pristine MP), incubated them withhuman plasma, and quantified the amount of adsorbed HSA byan ELISA (Figure 3c). We found that the absorbance level ofFigure 3. Human serum albumin capture efficiency using MP-PPEGMA-Ab in a whole human plasma solution measured by (a) anLangmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c04175Langmuir 2025, 41, 32432−3244232438https://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c04175/suppl_file/la5c04175_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175?fig=fig3&ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c04175?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asthe pristine MPs was approximately 30% lower than that of theCPB-modified counterpart (MP-PPEGMA-Ab), indicating thata smaller amount of HSA had been captured on the particlesurface. Without PPEGMA, nontargeted proteins in wholeplasma can be adsorbed to the surface, which hindered theinteraction between HSA and its complementary antibody dueto steric hindrance. As PPEGMA forms a hydrophilic coating,with the configuration of the CPB structure, we were able tominimize nonspecific protein interactions on the surface toincrease the sensitivity of our immunoassay. While it wasobserved that there was a stark difference between bothpristine MPs and MP-PPEGMA-Ab, further statistical analysisto validate the sensitivity differences was not performed due tothe limited sample size (n = 2). As it has been welldocumented that the presence of hydrophilic CPB coatingsreduces nonspecific protein adsorption, we believe that thesenoticeable differences were due to the presence of thePPEGMA coating. Therefore, due to the enhanced sensitivityimparted by the presence of the CPB, the utility of theseparticles could be used for detection of other clinicalapplications in which low-abundance proteins can be easilydetected without sample purification or pretreatment.78,794. CONCLUSIONIn this work, we designed and developed a highly sensitivemagnetic particle system for the capture and quantification ofdiagnostic marker proteins in clinical samples, such as serum orplasma. By using SI-ATRP, we were able to successfully graftconcentrated PPEGMA brushes onto the surface of theseparticles to minimize nonspecific protein absorption. Sub-sequently, we then modified these particles with a targetingantibody to specifically target HSA, where we were able toachieve a limit of detection as low as 6.4 ng mL−1 in unpurifiedwhole human plasma. From this, the use of CPBs showedexcellent potential for the development of ultrasensitivediagnostic tools in applications in highly abundant proteinenvironments.■ ASSOCIATED CONTENT*sı Supporting InformationThe Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c04175.Synthesis of PPEGMA-Br and PPEGMA-Azide, proteinadsorption test comparing CPB and SDPB, materialcharacterization of free PPEGMA-Br and MP-PPEGMA-Br, estimation of antibody functionalization, FTIR and1H NMR analysis of modified PPEGMA, SDS−PAGEand protein staining of MP-PPEGMA-Ab in a proteinsolution, and schematic of ELISAs (PDF)■ AUTHOR INFORMATIONCorresponding AuthorChiaki Yoshikawa − Research Center for Macromolecules andBiomaterials, National Institute for Materials Science(NIMS), Tsukuba, Ibaraki 305-0047, Japan; GraduateSchool of Life Science, Hokkaido University, Sapporo 060-0808, Japan; orcid.org/0000-0002-6589-387X;Phone: +81-29-860-4717; Email: YOSHIKAWA.Chiaki@nims.go.jpAuthorsGabriel Tai Huynh − Research Center for Macromoleculesand Biomaterials, National Institute for Materials Science(NIMS), Tsukuba, Ibaraki 305-0047, JapanJun Qiu − DSM Ahead/TS, 6167 DR Geleen, TheNetherlandsEdith van den Bosch − DSM Ahead/TS, 6167 DR Geleen,The NetherlandsTomohiko Yamazaki − Research Center for Macromoleculesand Biomaterials, National Institute for Materials Science(NIMS), Tsukuba, Ibaraki 305-0047, Japan; GraduateSchool of Life Science, Hokkaido University, Sapporo 060-0808, Japan; orcid.org/0000-0003-2136-8042Complete contact information is available at:https://pubs.acs.org/10.1021/acs.langmuir.5c04175Author ContributionsG.T.H.: writing of the original draft and review and editing.J.Q.: conceptualization, investigation, methodology, fundingacquisition, project administration, resources, and review andediting. E.v.d.B.: conceptualization, methodology, fundingacquisition, project administration, resources, and review andediting. T.Y.: data curation and review and editing. C.Y.:conceptualization, data curation, investigation, methodology,funding acquisition, project administration, resources, super-vision, writing of the original draft, and review and editing.NotesThe authors declare no competing financial interest.■ ACKNOWLEDGMENTSThis work was performed in part on the NIMS Molecular andMaterial Synthesis Platform. This work was partially supportedby JSPS KAKENHI Grants 19KK0368, 22H02133, and24KF0166.■ REFERENCES(1) Chen, Y.-T.; Kolhatkar, A. G.; Zenasni, O.; Xu, S.; Lee, T. R.Biosensing Using Magnetic Particle Detection Techniques. Sensors2017, 17 (10), 2300.(2) Avasthi, A.; Caro, C.; Pozo-Torres, E.; Leal, M. P.; García-Martín, M. L. Magnetic Nanoparticles as MRI Contrast Agents. InSurface-modified Nanobiomaterials for Electrochemical and BiomedicineApplications; Puente-Santiago, A. 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