Design And Synthesis Of Molecularly Imprinted Nanoparticles For The Diagnosis Of Diseases

Traditional diagnostic methods,such as immunoassays and nucleic acid-based assays,have been instrumental in disease detection,but they often face limitations in terms of sensitivity,specificity,and reproducibility.The design and synthesis of artificial nanomaterial-based antibodies hold immense importance in advancing various fields,from medicine to diagnostics and beyond.Here,we have designed and synthesized molecularly imprinted polymer（MIPs）based nanosized artificial antibodies for early diagnosis of diseases.These synthetic antibodies,often constructed using nanomaterials like nanoparticles or nanocomposites,offer a range of advantages over their natural counterparts.Their precise engineering allows for customization of binding affinities and specificities,enabling tailored recognition of target molecules such as disease biomarkers.Additionally,artificial nanomaterial-based antibodies exhibit superior stability and durability,providing resistance to harsh environmental conditions.The controlled synthesis of these nanomaterial-based antibodies allows for reproducibility and scalability,ensuring a stable supply for diverse applications.Their small size and unique properties make them ideal candidates for diagnostic platforms.Thus,the strategic design and synthesis of antibodies based on artificial nanomaterials contribute not only to the development of innovative technologies,but also hold the potential to revolutionize how we approach diagnostics,and therapy.I)A diverse library of nanoparticles ranging in size from 28-46 nm was synthesized for targeted interaction with the vimentin epitope peptide,vimentin and interfering protein,using a free radical polymerization.This strategic methodology not only allowed for the controlled fabrication of nanoparticles but also facilitated a systematic exploration of their properties,setting the foundation for a comprehensive understanding of their potential applications.Initial screening revealed nanoparticles with varying and notably high affinities for vimentin.Subsequently,the nanoparticle with high affinity for the vimentin epitope peptide(K_D=1.65×10^-10M)and vimentin(K_D=9.11×10-¹¹ M)was molecularly imprinted with the vimentin epitope peptide,resulting in nanomaterials demonstrating enhanced affinity(K_D=1.60×10-¹² M)and selectivity for the target protein.The study extended its investigation to evaluate the application of these molecularly imprinted nanoparticles in cancer cell lines Encouragingly,the results demonstrated promising results,highlighting the potential of these engineered nanoparticles as effective tools in cancer-related applications,with implications for targeted therapies and diagnostics.The incorporation of gold nanorods into nano MIP presented a groundbreaking strategy for precise cancer cell inactivation via photothermal treatment.This innovative study not only pioneers a new avenue to utilize nano MIP in cancer therapy but also underscores their potential application in the sensitive and specific detection of vimentin.The integration of gold nanorods not only enhances the therapeutic potential of nano MIP but also expands their utility to encompass targeted cancer treatment,thereby establishing a dual-functionality that contributes to the advancement of therapeutic and diagnostic approaches in oncology.II)Furthermore,we designed and applied molecularly imprinted nanoparticles（nano MIP）for the sensitive detection of C-reactive protein（CRP）using a solid-phase imprinting approach.Initially,the affinity of non-imprinted nanoparticles（nano NIP）was assessed against the epitope of CRP,CRP and other interfering proteins,revealing no significant differences in affinity.Subsequently,nano MIP were successfully imprinted with the epitope of CRP,leading to a remarkable two-order-of-magnitude increase in affinity towards CRP,while affinity towards other proteins remained minimal.The calculated limit of detection（LOD）for CRP was4.13 pg/m L,showcasing the high sensitivity of the proposed assay.Reproducibility tests showed impressive recovery rates ranging from 97.8%to 99.2%for CRP concentrations of 100,200,and 300 pg/m L,with a low relative standard deviation（RSD）between 0.65%and 0.88%.Notably,the RSD based on ten independently synthesized nano MIP@CDs stood at 1.8%,attesting to the exceptional reproducibility of the synthetic protocol.Furthermore,when applied to detect ten CRP serum samples with identical concentrations,the resulting RSD was 1.75%,affirming the commendable repeatability of this groundbreaking detection approach.Overall,the developed nano MIP@CDs demonstrate significant potential for precise and reproducible CRP detection in human serum samples.III)Also,we successfully engineered artificial synthetic antibodies targeting the cytotoxic T-lymphocyte-associated protein 4（CTLA-4）,a pivotal regulatory molecule crucial for orchestrating the immune system’s response to infections and cancer threats.Exploitation of the CTLA-4 pathway by certain cancer cells to avoid immune detection underscores the importance of developing targeted synthetic antibodies against this protein.To commence the synthesis,magnetic nanoparticles with a silica-coated layer were meticulously engineered,establishing a robust core-shell structure.Subsequently,the CTLA-4 epitope template was immobilized onto this solid support,serving as a foundation for the subsequent polymerization process.This methodology not only facilitated the creation of a molecularly imprinted polymer（nano MIP）with specificity toward the CTLA-4 epitope but also allowed the exploration of potential interference from other proteins.Bio-layer interferometry assay was employed to rigorously assess the binding affinity of both nano NIP,and nano MIP towards the CTLA4 epitope peptide,CTLA-4,and other proteins that could potentially interfere.The results of these assays provided crucial insight into the molecular recognition capabilities of the synthesized polymers.In particular,the affinity of nano MIP for the CTLA-4 protein was quantified at an impressive K_D=2.94×10^-10M,underscoring the high specificity,and selectivity achieved through the epitope imprinting process.This successful development of CTLA-4-specific nano MIP holds promise for the advancement of targeted therapeutic interventions and diagnostic applications in the context of cancer and immune-related disorders.In summary,nano MIP were synthesized using free radical polymerization for targeted interaction with the vimentin,CRP,and CTLA4 demonstrating high affinities.Molecular imprinting further enhanced the affinity as well as specificity of nano MIP.These nanoparticles showed promising results in cancer cell lines for targeted therapies and diagnostics,especially with the integration of gold nanorods for precise photothermal treatment.Additionally,nano MIP designed for sensitive detection of CRP,achieved a LOD of 4.13 pg/m L with high reproducibility and minimal interference.Synthetic antibodies targeting CTLA-4 were also developed,showing high specificity and potential for targeted cancer and immune therapy.