RESUMEN
Cell migration is an essential process of cancer metastasis. The spatiotemporal dynamics of signaling molecules influences cellular phenotypic outcomes. It has been increasingly documented that the Abelson (ABL) family kinases play critical roles in solid tumors. However, ABL1's shuttling dynamics in cell migration still remains unexplored. This is mainly because tools permitting the investigation of translocation dynamics of proteins in single living cells are lacking. Herein, to bridge this gap, we developed a unique multifunctional integrated single-cell analysis method that enables long-term observation of cell migration behavior and monitoring of signaling proteins and complexes at the subcellular level. We found that the shuttling of ABL1's to the cytoplasm results in a higher migration speed, while its trafficking back to the nucleus leads to a lower one. Furthermore, our results indicated that fluctuant protein-protein interactions between 14-3-3 and ABL1 modulate ABL1's nucleocytoplasmic fluctuation and eventually affect the cell speed. Importantly, based on these new insights, we demonstrated that disturbing ABL1's nuclear export traffic and 14-3-3-ABL1 complexes formation can effectively suppress cell migration. Thus, our method opens up a new possibility for simultaneous tracking of internal molecular mechanisms and cell behavior, providing a promising tool for the in-depth study of cancer.
Asunto(s)
Núcleo Celular , Neoplasias , Humanos , Núcleo Celular/metabolismo , Transporte Activo de Núcleo Celular , Proteínas/metabolismo , Citoplasma/metabolismo , Movimiento Celular , Neoplasias/metabolismoRESUMEN
Biomacromolecules exist and function in a crowded and spatially confined intracellular milieu. Single-cell analysis has been an essential tool for deciphering the molecular mechanisms of cell biology and cellular heterogeneity. However, a sound understanding of in vivo environmental effects on single-cell quantification has not been well established. In this study, via cell mimicking with giant unilamellar vesicles and single-cell analysis by an approach called plasmonic immunosandwich assay (PISA) that we developed previously, we investigated the effects of two in vivo environmental factors, i.e., molecular crowding and spatial confinement, on quantitative biochemistry in the cytoplasm of single cells. We find that molecular crowding greatly affects the biomolecular interactions and immunorecognition-based detection while the effect of spatial confinement in cell-sized space is negligible. Without considering the effect of molecular crowding, the results by PISA were found to be apparently under-quantitated, being only 29.5-50.0% of those by the calibration curve considering the effect of molecular crowding. We further demonstrated that the use of a calibration curve established with standard solutions containing 20% (wt) polyethylene glycol 6000 can well offset the effect of intracellular crowding and thereby provide a simple but accurate calibration for the PISA measurement. Thus, this study not only sheds light on how intracellular environmental factors influence biomolecular interactions and immunorecognition-based single-cell quantification but also provides a simple but effective strategy to make the single-cell analysis more accurate.
RESUMEN
Antibodies have been a mainstream class of therapeutics for clinical treatment of various diseases, especially cancers. However, mutation in cancer cells leads to resistance to therapeutic antibodies, hyperactivity of proliferation of cancer cells, and difficulty in the development of therapeutic antibodies. Herein, we present a strategy termed molecularly imprinted nanotransducer (MINT) for targeted photodynamic therapy (PDT) of mutated cancers. The MINT is a rationally engineered nanocomposite featuring a core of an upconversion nanoparticle, a shell of a thin layer of molecularly imprinted polymer, and a photosensitizer modified on the surface. As a proof-of-principle, truncated HER2 (P95HER2) overexpressed breast cancer, a challenging cancer lacking effective targeted therapeutics, was used as the cancer model. The designed structure, properties, functions, and anticancer efficacy of MINT were systematically investigated and experimentally confirmed. The MINT could not only specifically target P95HER2+ cancer cells in vitro and in vivo but also efficiently transfer the irradiated light and generate excited-state oxygen, resulting in efficient targeted cancer killing. Therefore, the MINT strategy provides a promising therapeutic for targeted PDT of drug-resistant cancers caused by target mutation.
Asunto(s)
Neoplasias de la Mama , Nanopartículas , Fotoquimioterapia , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Nanopartículas/química , Línea Celular TumoralRESUMEN
4'-Phosphopantetheinylation (4PPTylation) of proteins, which is derived from the hydrolysis of coenzyme A (CoA), is an essential post-translational modification participating in biosynthetic and metabolic pathways. However, due to the lack of specific recognition ligands as well as the shortage of sensitive analytical tools for single-cell analysis, the in-depth exploration of new cellular functions and mechanisms of protein 4PPTylation has been much hampered. In this study, we rationally engineered CoA-imprinted Raman nanotags for the specific recognition of 4PPTylation and thereby developed a molecularly imprinted polymer (MIP)-based plasmonic immunosandwich assay (PISA) for facile probing the 4PPTylation of ALDH1L1 in single cells. The molecularly imprinted nanotags exhibited excellent binding properties, giving a dissociation constant of 10-6 M and cross-reactivity values of less than 10%. The MIP-based PISA enabled the specific and sensitive detection of the level of 4PPTylated ALDH1L1 in single living cells. Particularly, monitoring of the fluctuation of 4PPTylated ALDH1L1 in single cells under simulation by an inhibitor (methotrexate) that acts on a different metabolism pathway was achieved, implying possible crosstalk between two different pathways in folate metabolism. Thus, the imprinted Raman nanotags-PISA provides a promising analytical tool with a single-cell resolution for exploring new functions and elucidating their mechanisms of protein 4PPTylation.
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Impresión Molecular , Polímeros , Polímeros/química , Proteínas , Polímeros Impresos Molecularmente , Oro/químicaRESUMEN
Phospholipids, as fundamental building blocks of the cell membrane, play important roles for molecule transportation, cell recognition, etc. However, due to the structural diversity and amphipathic nature, there are few methods for the specific recognition of lipids as compared to other biomolecules such as proteins and glycans. Herein, we developed a molecular imprinting strategy for controllable imprinting toward the polar head of phospholipid exposed on the surface of cellular membranes for recognition. Phosphatidylserine, as unique lipid on the outer membrane leaflet of exosome and also hallmark for cell apoptosis, was imprinted with the developed method. The phosphatidylserine imprinted materials showed high efficiency and specific targeting capability not only for apoptotic cell imaging but also for the isolation of exosomes. Collectively, the synthesized molecularly imprinted materials have great potential for selective plasma membrane recognition for targeted drug delivery and biomarker discovery.
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Impresión Molecular , Fosfolípidos , Epítopos/química , Fosfatidilserinas , Membrana Celular , Impresión Molecular/métodosRESUMEN
Harnessing innate immunity is an appealing strategy for cancer treatment. Herein, we report a new strategy called molecularly imprinted nanobeacons (MINBs) for redirecting innate immune killing towards triple-negative breast cancer (TNBC). The MINBs were molecularly imprinted nanoparticles with the N-epitope of glycoprotein nonmetastatic B (GPNMB) as the template and grafted with plentiful fluorescein moieties as the hapten. The MINBs could tag the TNBC cells via binding with GPNMB and thereby provide navigation for recruiting hapten-specific antibodies. The gathered antibodies could further trigger effective Fc-domain-mediated immune killing towards the tagged cancer cells. In vivo experiments showed that the TNBC growth was significantly inhibited after MINBs treatment by intravenous injection as compared with control groups. This study not only opens a new access for redirecting innate immunity towards TNBC but also paves the way for innate immunity-based therapy of other diseases.
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Nanopartículas , Neoplasias de la Mama Triple Negativas , Humanos , Anticuerpos , Línea Celular Tumoral , Glicoproteínas de Membrana , Impresión Molecular , Neoplasias de la Mama Triple Negativas/patología , Inmunidad InnataRESUMEN
Using two molecularly imprinted and cladded polymers (cMIPs), an inexpensive, fast and portable plasmonic immuno-sandwich assay (PISA) was rationally developed for high-specificity and ultra-sensitive detection of C-peptide in urine. The dual cMIPs-based PISA allowed healthy individuals to be distinguished from diabetes patients and exhibited several significant merits over existing immunoassays, holding great promise in clinical diagnosis.
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Impresión Molecular , Polímeros , Humanos , Inmunoensayo , PéptidosRESUMEN
Hypervalent bispecific gold nanoparticle-anchored aptamer chimeras (AuNP-APTACs) were designed as a new tool of lysosome-targeting chimeras (LYTACs) for efficient degradation of the ATP-binding cassette, subfamily G, isoform 2 protein (ABCG2) to reverse multidrug resistance (MDR) of cancer cells. The AuNP-APTACs could effectively increase the accumulation of drugs in drug-resistant cancer cells and provide comparable efficacy to small-molecule inhibitors. Thus, this new strategy provides a new way to reverse MDR, holding great promise in cancer therapy.
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Antineoplásicos , Nanopartículas del Metal , Oro/farmacología , Oro/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Resistencia a Antineoplásicos , Línea Celular Tumoral , Resistencia a Múltiples Medicamentos , Antineoplásicos/farmacologíaRESUMEN
Infectious virus diseases, particularly coronavirus disease 2019, have posed a severe threat to public health, whereas the developed therapeutic and prophylactic strategies are seriously challenged by viral evolution and mutation. Therefore, broad-spectrum inhibitors of viruses are highly demanded. Herein, an unprecedented antiviral strategy is reported, targeting the viral glycan shields with hypervalent mannose-binding nanoparticles. The nanoparticles exhibit a unique double-punch mechanism, being capable of not only blocking the virus-receptor interaction but also inducing viral aggregation, thereby allowing for inhibiting the virus entry and facilitating the phagocytosis of viruses. The nanoparticles exhibit potent and broad-spectrum antiviral efficacy to multiple pseudoviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its major variants (D614G, N501Y, N439K, Δ69-70, Delta, and Omicron; lentiviruses expressing only the spike proteins), as well as other vital viruses (human immunodeficiency virus 1 and Lassa virus), with apparent EC50 values around the 10-9 m level. Significantly, the broad-spectrum inhibition of authentic viruses of both wild-type SARS-CoV-2 and Delta variants is confirmed. Therefore, this hypervalent glycan-shield targeting strategy opens new access to broad-spectrum viral inhibition.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Antivirales/farmacología , Polisacáridos/metabolismoRESUMEN
Molecularly imprinted polymers (MIPs), as important mimics of antibodies, are chemically synthesized by polymerization in the presence of a target compound. MIPs have found wide applications in important fileds. However, the current molecular imprinting technology suffers from a dilemma; there is often a compromise between the best affinity and the best specificity for MIPs prepared under optimized conditions. Herein, we proposed a new strategy called molecular imprinting and cladding (MIC) to solve this issue. The principle is straightforward; after molecular imprinting, a chemically inert cladding thinlayer is generated to precisely cover non-imprinted area. We further proposed a special MIC approach for controllably engineering protein binders. The prepared cladded MIPs (cMIPs) exhibited significantly improved affinity and specificity. The general applicability of the proposed strategy and method was verified by engineering of cMIPs for the recognition of a variety of different proteins. The feasibility of cMIPs for real applications was demonstrated by fluorescence imaging of cancer cells against normal cells and immunoassay of C-peptide in human urine. This study opened up a new avenue for controllably engineering protein-specific antibody mimics with excellent recognition properties, holding great prospective in important applications such as disease diagnosis and nanomedicine.
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Impresión Molecular , Humanos , Impresión Molecular/métodos , Estudios Prospectivos , Anticuerpos/química , Proteínas , InmunoensayoRESUMEN
Blocking the PD-1/PD-L1 immune checkpoint has emerged as a promising strategy in cancer immunotherapy, in which monoclonal antibodies are predominately used as inhibitors. Despite their remarkable success, monoclonal antibody-based therapeutics suffer from drawbacks due to the use of antibodies, such as high cost, low stability and high frequency of immune-related adverse effects. Therefore, novel anti-PD-1/PD-L1 therapeutics that can address these issues are of significant importance. Herein, we report a molecularly imprinted polymer (MIP) based PD-1 nano inhibitor for blocking the PD-1/PD-L1 axis. The anti-PD-1 nanoMIP was rationally designed and engineered by epitope imprinting using the N-terminal epitope of PD-1 as the binding site. The anti-PD-1 nanoMIP showed good specificity and high affinity towards PD-1, yielding a disassociation constant at the 10-8 M level, much better than that between PD-1 and PD-L1. Via steric hindrance, this inhibitor could effectively block PD-1/PD-L1 interaction. Besides, it could effectively reactivate T cells and reverse the chemoresistance of tumor cells. Therefore, this present study not only provides a novel and promising immune checkpoint blockade inhibitor but also boosts further development of MIPs for cancer immunotherapy.
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Protein assays with fingerprints and high sensitivity are essential for biomedical research and applications. However, the prevailing methods mainly rely on indirect or labeled immunoassays, failing to provide fingerprint information. Herein, we report a dual biomimetic recognition-driven plasmonic nanogap-enhanced Raman scattering (DBR-PNERS) strategy for ultrasensitive protein fingerprinting and quantitation. A pair of molecularly imprinted nanoantennas were rationally engineered for specifically trapping a target protein into well-defined plasmonic nanogaps through dual-terminal recognition for ultrahigh Raman signal amplification. Meanwhile, a Raman-active small molecule was embedded into the nanoantenna as an internal standard to provide a ratiometric assay for robust quantitation. DBR-PNERS exhibited several significant merits over existing approaches, including fingerprinting, ultrahigh sensitivity, quantitation robustness, speed, sample consumption, and so on. Therefore, it can be a promising tool for a protein assay and holds a great perspective in important applications.
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Nanopartículas del Metal , Impresión Molecular , Espectrometría Raman , Oro , Biomimética , Mapeo Peptídico , ProteínasRESUMEN
Tyrosine phosphorylation is an important post-translational modification of proteins, and its accurate analysis is of vital importance. However, due to limited abundance of tyrosine phosphorylation as well as severe interference of serine/threonine phosphorylation and other phosphate-containing species, approaches that can directly analyse tyrosine phosphorylation on the cell membrane still remain limited. Herein, we report the rational development of molecularly imprinted and cladded Raman nanotags and their successful application in surface enhanced Raman spectroscopy (SERS) imaging of tyrosine phosphorylation on cancerous cells and tissues. The prepared molecularly imprinted and cladded SERS nanotags could specifically recognize phosphotyrosine and thereby allowed for distinguishing phosphotyrosine from other phosphate-containing species on cancerous cells and tissues by SERS imaging. Therefore, the molecularly imprinted and cladded nanotags-based SERS imaging can be a promising tool for tyrosine phosphorylation analysis and tyrosine phosphorylation-related studies, showing great potential for biomedical applications.
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Nanopartículas del Metal , Impresión Molecular , Impresión Molecular/métodos , Fosfotirosina , Fosforilación , Espectrometría Raman/métodos , Procesamiento Proteico-Postraduccional , Fosfatos , Nanopartículas del Metal/químicaRESUMEN
Chemodynamic therapy (CDT) that kills tumor cells by converting low-reactivity H2O2 into highly toxic hydroxyl radicals (â¢OH) is an emerging tumor therapeutic modality, but its therapeutic efficacy is largely limited by both the lack of tumor targeting and redox homeostasis in tumor cells. Herein, we report Cu2+-encapsulated and GalNAc-imprinted biodegradable silica nanoparticles (nanoMIP) for boosting CDT. In this strategy, the Cu2+ was first encapsulated into disulfide-bridged silica nanoparticles with a high loading capacity of â¼18.3%, followed by in situ functionalization via molecular imprinting using GalNAc as a template. Such a nanovector could specifically target tumor cells overexpressing the Tn antigen to promote the cellular uptake. After internalization into tumor cells, the degradation of nanoMIP occurred in response to the tumor microenvironment, spontaneously releasing Cu2+/Cu+ via redox cycles, which in turn promoted highly potent GSH depletion and triggered â¢OH generation by a Fenton-like reaction. Notably, we found that the catalase activity could be effectively inhibited by the produced Cu+, which indirectly upregulated the endogenous H2O2 level. As a result, the "maladjusted" tumor cells lost the resistance against â¢OH damage, finally resulting in the apoptosis of tumor cells. In vitro and in vivo experiments demonstrated that our nanoMIP exhibited excellent cytotoxicity against tumor cells and high efficacy of tumor inhibition in the xenograft tumor model with negligible side effects. Taken together, our study provides not only a promising strategy for maximizing the CDT efficacy but also a new insight for developing MIP-based nanomedicine.
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Nanopartículas , Neoplasias , Catalasa/metabolismo , Línea Celular Tumoral , Disulfuros/farmacología , Homeostasis , Humanos , Peróxido de Hidrógeno/metabolismo , Nanopartículas/uso terapéutico , Neoplasias/terapia , Oxidación-Reducción , Dióxido de Silicio/farmacología , Microambiente TumoralRESUMEN
Nanozymes have attracted wide attention for the unique advantages of low cost, high stability and designability. Molecularly imprinted polymers (MIPs) have demonstrated great potential as a new type of nanozymes due to their excellent specificity and high affinity. However, effective approaches for creating molecularly imprinted nanozymes still remain limited. Herein, reverse microemulsion template docking surface imprinting (RMTD-SI) is reported as a new approach for the rational design and engineering of nanozymes with free substrate access for the ligation of ssDNA sequences. As a proof of the principle, octa-deoxyribonucleotide-imprinted nanoparticles were successfully prepared. Using tetradeoxyribonucleotides and octa-deoxyribonucleotide as substrates, the properties, catalytic activity and behavior of the imprinted nanoparticles were thoroughly investigated. The imprinted nanozyme exhibited an enhanced reaction speed (by up to 41-fold) and good sequence selectivity towards substrate tetra-deoxyribonucleotides. More interestingly, due to the open substrate access, the imprinted nanozyme also allowed the ligation of a ssDNA that fully matched with the imprinted cavity and other ssDNA substrates to form longer sequences, but at the price of substrate selectivity. Thus, this study provides not only a new avenue to the rational design and synthesis of molecularly imprinted nanozymes but also new insights of their catalytic behavior.
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Impresión Molecular , Nanopartículas , Polímeros/química , DesoxirribonucleótidosRESUMEN
A new method called reverse microemulsion-confined ganglioside-oriented surface imprinting and cladding (RM-GOSIC) is presented for controllable preparation of nanoscale binders for high-affinity targeting gangliosides. Using GM1a, an affordable ganglioside, as a representative ganglioside target, single-core quantum dot GM1a-imprinted and GM1a-cladded polymer (cMIP) nanoparticles were prepared. The prepared cMIP nanoparticles exhibited extremely high affinity towards GM1a, with dissociation constant at the nanomolar level (3-6 nM). The prepared cMIP nanoparticles also recognized structurally closed gangliosides while their cross-reactivity towards other gangliosides remained low. The potential of the cMIP nanoparticles in biomedical applications was demonstrated by cell and tissue imaging. Thus, this approach opened a new access to the synthesis of high-affinity nanoscale binders for targeting gangliosides.
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Nanopartículas , Puntos Cuánticos , Gangliósidos , PolímerosRESUMEN
The ATP-binding cassette, subfamily G, isoform 2 protein (ABCG2), as an important member of ABC transporters, plays a key role in multidrug resistance (MDR) in cancer and has been widely considered as a marker of cancer stem cells (CSC). Reagents capable of simultaneously targeting ABCG2 and reversing MDR have great clinical application values, but their development is highly challenging. Herein, ABCG2 glycosylated extracellular region-binding aptamers were efficiently screened by a cladded molecularly imprinted polymer (cMIP)-based in vitro screening method and further rationally engineered into cyclic bivalent aptamers. Experiments showed that both the monovalent and cyclic bivalent aptamers could specifically bind ABCG2 and thereby specially target CSC of human colorectal carcinomas (CoCSC), while the latter could effectively reverse MDR in drug-resistant liver cancer cells (HepG2/ADR). Different from currently predominant small molecule inhibitors, the reversal of MDR relied on a different mechanism; the cyclic bivalent aptamers bound the two monomers of ABCG2 dimers simultaneously and thereby blocked the ABCG2-mediated drug-pumping channel, resulting in increased intracellular accumulation of substrate drugs. This study opened a new access to the development of affinity reagents for targeting CSC and reversing MDR, holding great prospects in cancer diagnosis and treatment.
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Antineoplásicos , Neoplasias , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Humanos , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismoRESUMEN
Enzyme mimics are of significant importance due to their facile preparation, low cost and stability to rigorous environments. Molecularly imprinted polymers (MIPs) have been important synthetic mimics of enzymes. However, effective strategies for the rational design of enzyme-mimicking MIPs have still remained limited. Herein, we report a new strategy, termed affinity gathering-enhanced coupling and thermal cycling amplification (AGEC-TCA), for the rational design and engineering of molecularly imprinted mesoporous silica nanoparticles (MSNs) that are capable of ligating short ssDNA fragments. This strategy relied on enhancing the effective collision probability via binding substrates into highly favorable orientation by product-imprinted MSNs as well as product release via thermal cycling which enabled successive product amplification. Using modified and natural hexadeoxyribonucleotide as templates, the prepared product-imprinted MSNs exhibited a remarkably enhanced reaction speed (by up to 63-fold) as well as excellent sequence specificity towards substrate trideoxyribonucleotides. Thus, this strategy opened up a new avenue to access enzyme mimics via molecular imprinting.
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Impresión Molecular , Nanopartículas , ADN Ligasas , Polímeros Impresos Molecularmente , Nanopartículas/química , Dióxido de SilicioRESUMEN
Nanoparticles have been widely used in important biomedical applications such as imaging, drug delivery, and disease therapy, in which targeting toward specific proteins is often essential. However, current targeting strategies mainly rely on surface modification with bioligands, which not only often fail to provide desired properties but also remain challenging. Here an unprecedented approach is reported, called reverse microemulsion-confined epitope-oriented surface imprinting and cladding (ROSIC), for facile, versatile, and controllable engineering coreless and core/shell nanoparticles with tunable monodispersed size as well as specific targeting capability toward proteins and peptides. Via engineering coreless imprinted and cladded silica nanoparticles, the effectiveness and superiority over conventional imprinting of the proposed approach are first verified. The prepared nanoparticles exhibit both high specificity and high affinity. Using quantum dots, superparamagnetic nanoparticles, silver nanoparticles, and upconverting nanoparticles as a representative set of core substrates, a variety of imprinted and cladded single-core/shell nanoparticles are then successfully prepared. Finally, using imprinted and cladded fluorescent nanoparticles as probes, in vitro targeted imaging of triple-negative breast cancer (TNBC) cells and in vivo targeted imaging of TNBC-bearing mice are achieved. This approach opens a new avenue to engineering of nanoparticles for targeting specific proteins, holding great prospects in biomedical applications.
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Neoplasias de la Mama/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas de Magnetita/química , Impresión Molecular/métodos , Animales , Modelos Animales de Enfermedad , Nanopartículas del Metal/química , Ratones , Puntos Cuánticos/química , Dióxido de Silicio/química , Plata/químicaRESUMEN
Phosphorylation is one of the most frequently occurring post-translation modifications in mammals. Because abnormal protein phosphorylation is related to many diseases, phosphorylation analysis is essential for a sound understanding of protein phosphorylation and its relationship with diseases. Among several types of reagents for phosphorylation recognition, molecularly imprinted polymers (MIPs), as synthetic mimics of antibodies, have exhibited unique strengths that can overcome the drawbacks of biological reagents. However, the performance of current MIPs has remained unideal. Meanwhile, while the currently existing imprinting methods have permitted the production of several material formats, such as crushed particles and mesoporous nanoparticles, a general method allowing for the preparation of monodispersed molecularly imprinted nanoparticles has not been developed yet. Herein, we report a new approach called reverse microemulsion template docking surface imprinting and cladding (RMTD-SIC) for facile preparation of monodispersed imprinted nanoparticles for better phosphorylation recognition. Through rational design and controllable engineering, monodisperse imprinted and cladded nanoparticles specific to general phosphorylation and tyrosine phosphorylation were synthesized, which yield the highest imprinting factors as compared with published studies. The prepared nanomaterials exhibited excellent specificity and affinity, allowing for specific enrichment and improved mass spectrometric identification of target phosphorylated peptides from complex samples containing 100-fold more abundant interfering peptides. Therefore, the RMTD-SIC approach holds great potential for phosphorylation analysis and phosphorylation recognition-based applications.