Facile biotic/abiotic sandwich detection system for the highly sensitive detection of human serum albumin and glycated albumin.

Quantifying glycated albumin (GA) levels in the blood is crucial for diagnosing diabetes because they strongly correlate with blood glucose concentration. In this study, a biotic/abiotic sandwich assay was developed for the facile, rapid, and susceptible detection of human serum albumin (HSA) and GA. The proposed sandwich detection system was assembled using a combination of two synthetic polymer receptors and natural antibodies. Molecularly imprinted polymer nanogels (MIP-NGs) for HSA (HSA-MIP-NGs) were used to mimic capture antibodies, whereas antibodies for HSA or GA were used as primary antibodies and fluorescent signaling MIP-NGs for the Fc domain of IgG (F-Fc-MIP-NGs) were used as a secondary antibody mimic to indicate the binding events. The HSA/anti-HSA/F-Fc-MIP-NGs complex, formed by incubating HSA and anti-HSA antibodies with F-Fc-MIP-NGs, was captured by HSA-MIP-NGs immobilized on the chips for fluorescence measurements. The analysis time was less than 30 min, and the limit of detection was 15 pM. After changing the anti-HSA to anti-GA (monoclonal antibody), the fluorescence response toward GA exceeded that of HSA, indicating successful GA detection using the proposed sandwich detection system. Therefore, the proposed system could change the detection property by changing a primary antibody, indicating that this system can be applied to various target proteins and, especially, be a powerful approach for facile and rapid analysis methods for proteins with structural similarity.

Protein-imprinted polymer films prepared via cavity-selective multi-step post-imprinting modifications for highly selective protein recognition.

The use of molecularly imprinted polymers (MIPs) for achieving synthetic receptors capable of selective molecular recognition is promising; however, these polymers exhibit low selectivity derived from the heterogeneity of their created, imprinted cavities. To achieve highly selective protein recognition, we herein report the cavity-selective, multi-step, post-imprinting modification of MIPs. An MIP film for lysozyme was prepared by the copolymerization of {[2-(2-methacrylamido)ethyldithio]ethylcarbamoyl}methoxy acetic acid, a functional monomer possessing a modifiable disulfide bond, with acrylamide and N,N'-methylenebisacrylamide in the presence of lysozyme. After the removal of lysozyme, the disulfide bonds were cleaved by treatment with a reductant. A low concentration of lysozyme was then added to occupy the high-affinity cavities of the polymer and sterically protect the thiol groups within them. A poly(ethylene glycol)-based capping agent was reacted with the thiol groups residing in low-affinity cavities to hinder them. After the regeneration of the high-affinity cavities by washing out the bound lysozyme, the remaining thiol groups were reacted with 3-(2-pyridyldithio)propionic acid to introduce interacting groups, which produced capped MIPs. Comparing the capped and uncapped MIPs revealed that off-target protein binding was effectively suppressed by the capping treatment without any reduction in binding affinity (1.1 × 109 M-1). Further investigation revealed that the lysozyme concentration during the capping process is critical for the selectivity of the capped MIP. In this case, highly selective MIPs were achieved when the lowest lysozyme concentration (100 nM) was used. This facile process for creating highly selective, synthetic polymer receptors is a powerful approach for achieving plastic antibodies.

Antibody-Conjugated Signaling Nanocavities Fabricated by Dynamic Molding for Detecting Cancers Using Small Extracellular Vesicle Markers from Tears.

Small extracellular vesicles (sEVs) are reliable biomarkers for early cancer detection; however, conventional detection methods such as immune-based assays and microRNA analyses are not very sensitive and require sample pretreatments and long analysis time. Here, we developed a molecular imprinting-based dynamic molding approach to fabricate antibody-conjugated signaling nanocavities capable of size recognition. This enabled the establishment of an easy-to-use, rapid, sensitive, pretreatment-free, and noninvasive sEV detection platform for efficient sEV detection-based cancer diagnosis. An apparent dissociation constant was estimated to be 2.4 × 10-16 M, which was ∼1000 times higher than that of commercial immunoassays (analysis time, 5 min/sample). We successfully used tears for the first time to detect cancer-related intact sEVs, clearly differentiating between healthy donors and breast cancer patients, as well as between samples collected before and after total mastectomy. Our nanoprocessing strategy can be easily repurposed for the specific detection of other types of cancer by changing the conjugated antibodies, thereby facilitating the establishment of liquid biopsy for early cancer diagnosis.

Molecularly Imprinted Nanogels Capable of Porcine Serum Albumin Detection in Raw Meat Extract for Halal Food Control.

Accurate, simple, and valuable analytical methods for detection of food contamination are rapidly expanding to evaluate the validity of food product quality because of ethnic considerations and food safety. Herein molecularly imprinted nanogels (MIP-NGs), capable of porcine serum albumin (PSA) recognition, were prepared as artificial molecular recognition elements. The MIP-NGs were immobilized on a quartz crystal microbalance (QCM) sensor for detection of pork contamination in real beef extract samples. The MIP-NGs-based QCM sensor showed high affinity and excellent selectivity toward PSA compared to reference serum albumins from five different animals. The high PSA specificity of MIP-NGs led to the detection of pork contamination with a detection limit of 1% (v/v) in real beef extract samples. We believe the artificial molecular recognition materials prepared by molecular imprinting are a promising candidate for halal food control.

Molecularly Imprinted Nanogels Possessing Dansylamide Interaction Sites for Controlling Protein Corona In Situ by Cloaking Intrinsic Human Serum Albumin.

Nanomaterials have become increasingly promising for biomedical applications owing to their specific biological characteristics. As drug delivery vehicles, nanomaterials have to circulate in the bloodstream to deliver the encapsulated components to the target tissues. Protein corona regulation is one of the promising approaches that gives stealth capability to avoid immune response. The aim of this study was to develop molecularly imprinted polymer nanogels (MIP-NGs) capable of protein corona regulation, using intrinsic human serum albumin (HSA) and with a functional monomer, dansylamide ethyl acrylamide (DAEAm), the dansylamide group serving as a ligand for HSA. The recognition capability of HSA for MIP-NGs was investigated by isothermal titration calorimetry (ITC). The affinity of the MIP-NGs prepared with DAEAm was then compared to that of the reference MIP-NGs prepared with pyrrolidyl acrylate developed in our previous study. Furthermore, we demonstrated that the concurrent use of these two different functional monomers for molecular imprinting was further effective to construct high-affinity recognition nanocavities for HSA and to form HSA-rich protein corona in the human plasma owing to the different interaction modes of the monomers. We believe that the molecular imprinting strategy developed through the use of ligand-based functional monomer is an effective strategy to create artificial molecular recognition materials.

Orientationally Fabricated Zwitterionic Molecularly Imprinted Nanocavities for Highly Sensitive Glycoprotein Recognition.

Glycoprotein recognition has recently gained a lot of attention, since glycoproteins play important roles in a diverse range of biological processes. Robustly synthesized glycoprotein receptors, such as molecularly imprinted polymers (MIPs), which can be easily and sustainably handled, are highly attractive as antibody substitutes because of the difficulty in obtaining high-affinity antibodies specific for carbohydrate-containing antigens. Herein, molecularly imprinted nanocavities for glycoproteins have been fabricated via a bottom-up molecular imprinting approach using surface-initiated atom transfer radical polymerization (SI-ATRP). As a model glycoprotein, ovalbumin was immobilized in a specific orientation onto a surface plasmon resonance sensor chip by forming a conventional cyclic diester between boronic acid and cis-diol. Biocompatible polymer matrices were formed around the template molecule, ovalbumin, using SI-ATRP via a hydrophilic comonomer, 2-methacryloyloxyethyl phosphorylcholine, in the presence of pyrrolidyl acrylate (PyA), a functional monomer capable of electrostatically interacting with ovalbumin. The removal of ovalbumin left MIPs with binding cavities containing boronic acid and PyA residues located at suitable positions for specifically binding ovalbumin. Careful analysis revealed that strict control over the polymer significantly improved sensitivity and selectivity for ovalbumin recognition, with a limit of detection of 6.41 ng/mL. Successful detection of ovalbumin in an egg white matrix was demonstrated to confirm the practical utility of this approach. Thus, this strategy of using a polymer-based recognition of a glycoprotein through molecularly imprinted nanocavities precisely prepared using a bottom-up approach provides a potentially powerful approach for detection of other glycoproteins.

Site-specific post-imprinting modification of molecularly imprinted polymer nanocavities with a modifiable functional monomer for prostate cancer biomarker recognition.

Recognition of glycans of glycoproteins using biotic materials such as antibodies is challenging due to lack of antigenicity. Polymeric materials suitable for the molecular recognition of glycoproteins have attracted considerable attention. In this study, we aimed to develop abiotic molecular materials for the recognition of prostate-specific antigen (PSA), a known biomarker for prostate cancer. We used a non-covalent bonding-based molecular imprinting technique to introduce post-imprinting poly(ethylene glycol)-based capping agent into a low-affinity recognition cavity. Details of the binding properties of these groups were investigated to optimize their affinity and selectivity for PSA. Molecularly imprinted polymers (MIPs) were prepared using a bottom-up approach based on surface-initiated atom transfer radical polymerization from a PSA-conjugated sensor chip with a functional monomer-bearing carboxy and secondary amine groups as interaction and post-imprinting modification (PIM) sites, respectively. PSA was orientationally conjugated on the sensor chip through diesters between the immobilized 3-fluorophenyl boronic acid and the cis-diol groups of PSA glucans. Treatment with the capping agent selectively inactivated low-affinity recognition cavities while protecting high-affinity cavities with the addition of a low concentration of PSA as a dynamic protection agent. The MIP thickness is critical in the present molecular imprinting, as a value of less than 5 nm can enable high selectivity. We believe that the proposed strategy based on a non-covalent molecular imprinting approach combined with a PIM-based capping treatment provides a novel method for the development of highly sensitive and selective glycoprotein recognition materials for use in biomarker sensing.

A Pretreatment-Free, Polymer-Based Platform Prepared by Molecular Imprinting and Post-Imprinting Modifications for Sensing Intact Exosomes.

Exosomes are small (30-100 nm) membrane vesicles that serve as regulatory agents for intercellular communication in cancers. Currently, exosomes are detected by immuno-based assays with appropriate pretreatments like ultracentrifugation and are time consuming (>12 h). We present a novel pretreatment-free fluorescence-based sensing platform for intact exosomes, wherein exchangeable antibodies and fluorescent reporter molecules were aligned inside exosome-binding cavities. Such antibody-containing fluorescent reporter-grafted nanocavities were prepared on a substrate by well-designed molecular imprinting and post-imprinting modifications to introduce antibodies and fluorescent reporter molecules only inside the binding nanocavities, enabling sufficiently high sensitivity to detect intact exosomes without pretreatment. The effectiveness of the system was demonstrated by using it to discriminate between normal exosomes and those originating from prostate cancer and analyze exosomes in tear drops.

Regulation of protein-binding activities of molecularly imprinted polymers via post-imprinting modifications to exchange functional groups within the imprinted cavity.

We prepared lysozyme-imprinted polymers bearing modifiable sites within the imprinted cavity to introduce various functional groups via post-imprinting modifications. For this purpose, ({[2-(2-methacrylamido)-ethyldithio]-ethylcarbamoyl}-methoxy)acetic acid (MDTA), which has a carboxy group to interact with the target protein, lysozyme, and a disulfide linkage for post-imprinting modifications, was used as a functional monomer. A lysozyme-imprinted polymer film was prepared by copolymerization of MDTA with a cross-linker, N,N'-methylenebisacrylamide, in the presence of lysozyme. After removing lysozyme, followed by reducing the disulfide linkage, various functional groups, such as carboxy, amino, sulfonate, and oligo-ethylene oxide, were introduced to the exposed thiol groups via a disulfide exchange reaction using the pyridyldisulfide derivatives of these functional groups. Various functional groups could be introduced reversibly via this post-imprinting disulfide exchange reaction after the construction of the lysozyme-imprinted cavities. The modification regulated the protein-binding activity. The proposed post-imprinting modification system, based on a molecular imprinting process, is expected to lead to the development of advanced materials for fine-tuning and/or introducing desired functions.

Regioselective Molecularly Imprinted Reaction Field for [4 + 4] Photocyclodimerization of 2-Anthracenecarboxylic Acid.

Molecularly imprinted cavities have functioned as a regioselective reaction field for the [4 + 4] photocyclodimerization of 2-anthracenecarboxylic acid (2-AC). Molecularly imprinted polymers were prepared by precipitation polymerization of N-methacryloyl-4-aminobenzamidine as a functional monomer to form a complex with template 2-AC and ethylene glycol dimethacrylate as a crosslinking monomer. The 2-AC-imprinted cavities thus constructed preferentially bound 2-AC with an affinity greater than that toward structurally related 9-anthracenecarboxylic acid, 2-aminoanthracene, and unsubstituted anthracene. Moreover, from the four possible regioisomeric cyclodimers, they mediated the [4 + 4] photocyclodimerization of 2-AC specifically to the anti-head-to-tail (anti-HT) isomer. This indicates that the imprinted cavities accommodate two 2-AC molecules in an anti-HT manner, thereby facilitating the subsequent regioselective photocyclodimerization.

A Programmable Signaling Molecular Recognition Nanocavity Prepared by Molecular Imprinting and Post-Imprinting Modifications.

Inspired by biosystems, a process is proposed for preparing next-generation artificial polymer receptors with molecular recognition abilities capable of programmable site-directed modification following construction of nanocavities to provide multi-functionality. The proposed strategy involves strictly regulated multi-step chemical modifications: 1) fabrication of scaffolds by molecular imprinting for use as molecular recognition fields possessing reactive sites for further modifications at pre-determined positions, and 2) conjugation of appropriate functional groups with the reactive sites by post-imprinting modifications to develop programmed functionalizations designed prior to polymerization, allowing independent introduction of multiple functional groups. The proposed strategy holds promise as a reliable, affordable, and versatile approach, facilitating the emergence of polymer-based artificial antibodies bearing desirable functions that are beyond those of natural antibodies.

Preparation of molecularly imprinted polymers for the recognition of proteins via the generation of peptide-fragment binding sites by semi-covalent imprinting and enzymatic digestion.

Molecularly imprinted polymers bearing peptide fragment-based binding sites within the protein-imprinted cavities were prepared by copolymerization of the acrylated protein with 6-monoacryloyl-trehalose and 6,6'-diacryloyl-trehalose as a hydrophilic comonomer and a crosslinker respectively, followed by enzymatic decomposition of the grafted protein into the polymer matrix with pepsin, resulting in the creation of peptide fragment-based protein-binding sites.

Conjugated-protein mimics with molecularly imprinted reconstructible and transformable regions that are assembled using space-filling prosthetic groups.

Conjugated-protein mimics were obtained using a new molecular imprinting strategy combined with post-imprinting modifications. An antibiotic was employed as a model template molecule, and a polymerizable template molecule was designed, which was composed of the antibiotic and two different prosthetic groups attached through a disulfide bond and Schiff base formation. After co-polymerization with a cross-linker, the template molecule was removed together with the prosthetic groups, yielding the apo-type scaffold. Through conjugation of the two different prosthetic groups at pre-determined positions within the apo-type scaffold, the apo cavity was transformed into a functionalized holo cavity, which enables the on/off switching of the molecular recognition ability, signal transduction activity for binding events, and photoresponsive activity.

A rapid abiotic/biotic hybrid sandwich detection for trace pork adulteration in halal meat extract.

In this study, we prepared molecularly imprinted polymer nanogels with good affinity for the Fc domain of immunoglobulin G (IgG) using 4-(2-methacrylamidoethylaminomethyl) phenylboronic acid as a modifiable functional monomer for post-imprinting in-cavity modification of a fluorescent dye (F-Fc-MIP-NGs). A novel nanogel-based biotic/abiotic hybrid sandwich detection system for porcine serum albumin (PSA) was developed using F-Fc-MIP-NGs as an alternative to a secondary antibody for fluorescence detection and another molecularly imprinted polymer nanogel capable of recognizing PSA (PSA-MIP-NGs) as a capturing artificial antibody, along with a natural antibody toward PSA (Anti-PSA) that was used as a primary antibody. After incubation of PSA and Anti-PSA with F-Fc-MIP-NGs, the PSA/Anti-PSA/F-Fc-MIP-NGs complex was captured by immobilized PSA-MIP-NGs for fluorescence measurements. The analysis time was less than 30 min for detecting pork adulteration of 0.01 wt% in halal beef and lamb meats. The detection limit was comparable to that of frequently used immunoassays. Therefore, we believe that this method is a promising, sensitive, and rapid detection method for impurities in real samples and could be a simple, inexpensive, and rapid alternative to conventional methods that have cumbersome procedures of 4 hours or more.

A molecularly imprinted nanocavity with transformable domains that fluorescently indicate the presence of antibiotics in meat extract samples.

In this study, we aimed to create synthetic polymer receptors with the fluorescence signalling ability, using molecular imprinting, precisely designed template molecules, and site-specific post-imprinting modifications, which can mimic conjugated proteins and are capable of specific molecular recognition, and wherein successful binding can be indicated by a change in fluorescence. A molecularly imprinted APO-type nanocavity with a reconstructable domain was prepared by co-polymerisation of a template molecule containing cephalexin conjugated to polymerisable groups via a Schiff base, a disulphide bond, and a cross-linker, followed by hydrolysis of the Schiff base and a disulphide exchange reaction. Fluorescence-based indication of binding was devised by the Schiff base formation reaction with 4-formylsalicylic acid, and the interacting site was introduced via a disulphide exchange reaction with 4-mercaptobenzoic acid, yielding a multifunctional mature (HOLO)-type molecularly imprinted nanocavity. The ability to indicate binding events using changes in the fluorescence of the HOLO polymer was investigated, and it was revealed that the target antibiotic cephalexin can be selectively detected in aqueous media with high affinity (Ka = 1.1 × 104 M-1). Furthermore, the proposed sensor exhibited the potential to detect spiked cephalexin in chicken extracts with a limit of detection of 18 µM (1.3 ppm). The proposed fluorescence-sensing system based on molecular imprinting and post-imprinting modification is expected to enable the development of advanced materials for the specific detection of trace antibiotics in complex samples.

Fc Domain-Imprinted Stealth Nanogels Capable of Orientational Control of Immunoglobulin G Adsorbed In Vivo.

Regulation of nanomaterial-cell interaction is an important requisite for a variety of biomedical applications such as drug delivery systems and theranostics. Here, we demonstrate the regulation of nanomaterial-cell interaction using the oriented adsorption of intrinsic immunoglobulin G (IgG) on molecularly imprinted polymer nanogels (MIP-NGs) capable of recognizing the fragment crystallizable (Fc) domain of IgG. The unique domain recognition property resulted in the suppression of the immune response in Fc domain receptor-possessing macrophages and natural killer cells due to the regulation of protein corona based on the oriented adsorption of IgG. This resulted in the hindrance of the Fc domain, which is the trigger of an immune response. Furthermore, the acquisition of stealth capability was successfully demonstrated in vivo using intravital confocal laser scanning microscopy. The domain imprinting proposed in this study will provide a new strategy for creating nanomaterials capable of domain recognition-based oriented adsorption of intrinsic proteins in situ, thus regulating the protein corona formed on the nanomaterials. Thus, the unique Fc domain-recognition nanomaterial developed in our study can be used for various biomedical applications to target specific cells without triggering an immune response.

Biocompatible polymer-modified gold nanocomposites of different shapes as radiation sensitizers.

Radiation therapy is a powerful approach for cancer treatment due to its low invasiveness. The development of radiation sensitizers is of great importance as they assist in providing radiation therapy at a low dose. In this study, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC)-modified gold nanocomposites of different shapes were created using the grafting-to approach to serve as a novel radiation sensitizer with high cellular uptake. The effect of the shape of the nanocomposite on cellular uptake by the breast cancer cell line MCF-7 was also investigated. The PMPC-modified gold nanostars showed the highest cellular uptake compared to the other gold nanocomposites (spheres and rods), whereas cell cytotoxicity was negligible among all candidates. Furthermore, the therapeutic effect of radiation of PMPC-modified nanostars was the highest among all the gold nanocomposites. These results clearly indicate that the shape of the gold nanocomposite is an important parameter for cellular uptake and radiation sensitizing effects in breast cancer cells.

In vivo stealthified molecularly imprinted polymer nanogels incorporated with gold nanoparticles for radiation therapy.

Radiation therapy is a representative therapeutic approach for cancer treatment, wherein the development of efficient radiation sensitizers with low side effects is critical. In this study, a novel stealth radiation sensitizer based on Au-embedded molecularly imprinted polymer nanogels (Au MIP-NGs) was developed for low-dose X-ray radiation therapy. Surface plasmon resonance measurements reveal the good affinity and selectivity of the obtained Au MIP-NGs toward the target dysopsonic protein, human serum albumin. The protein recognition capability of the nanogels led to the formation of the albumin-rich protein corona in the plasma. The Au MIP-NGs acquire stealth capability in vivo through protein corona regulation using the intrinsic dysopsonic proteins. The injection of Au MIP-NGs improved the efficiency of the radiation therapy in mouse models of pancreatic cancer. The growth of the pancreatic tumor was inhibited even at low X-ray doses (2 Gy). The novel strategy reported in this study for the synthesis of stealth nanomaterials based on nanomaterial-protein interaction control shows significant potential for application even in other approaches for cancer treatment, diagnostics, and theranostics. This strategy paves a way for the development of a wide range of effective nanomedicines for cancer therapy.

Molecularly imprinted polymer nanogel-based fluorescence sensing of pork contamination in halal meat extracts.

Pork contamination is a serious concern for the global halal food market because many manufacturers commonly use pork instead of beef to reduce production costs. In this study, a highly sensitive fluorescent molecularly imprinted polymer nanogel (F-MIP-NG)-based sensor was developed for rapid porcine serum albumin (PSA) detection to investigate pork contamination in halal meat extracts. F-MIP-NGs were prepared via molecular imprinting and conjugation with ATTO 647N as the fluorescent reporter molecule for the post-imprinting modification (PIM) and then immobilized on gold-coated sensor chips. For achieving rapid and easy measurement, the fluorescence response was measured using a custom-made liquid handling robot equipped with a fluorescence microscope. The fluorescence response increased with increasing PSA concentration. Under optimal conditions, the F-MIP-NG-based sensors exhibited high sensitivity, a detection limit of 40 pM, a linear range of 0.25-5 nM, and excellent affinity and selectivity towards PSA, compared to potentially interfering proteins. Moreover, it was more efficient to detect beef contamination in 1 wt% pork contamination compared to the real-time polymerase chain reaction. Collectively the good analytical performance, high rates of recovery in real meat extract samples, fast detection, and a low detection limit of pork contamination (0.1 wt%) indicated the potential of the proposed sensor for detecting PSA as a marker of pork contamination in halal meat samples. The proposed sensing system based on the MIPs would open a way to establish highly sensitive and rapid sensing systems (<5 min/sample) for food analysis.

Signalling molecular recognition nanocavities with multiple functional groups prepared by molecular imprinting and sequential post-imprinting modifications for prostate cancer biomarker glycoprotein detection.

Fluorescent-signalling molecularly-imprinted nanocavities possessing orthogonal dual interaction sites for the detection of prostate cancer biomarker glycoprotein were constructed through molecular imprinting and sequential multistep post-imprinting modifications (PIMs) using a newly designed multi-functionalised PIM reagent (PIR). The PIR, possessing an interaction site and dual reaction sites for PIMs, enabled us to introduce multiple functions including interaction sites and fluorescent reporter groups in a single PIM site, leading to the sensitive fluorescent detection of target glycoproteins with a high signal-to-noise ratio. Prostate specific antigen (PSA), used as a biomarker for prostate-related diseases, was selected as a target glycoprotein. Surface-initiated atom transfer radical polymerisation from template PSA immobilised the substrate with a functional monomer possessing a phenyl boronic acid group, where the template PSA was designed to possess polymerisation groups aligned with disulphide linkage. Using the thiol groups left after removing templates, PIR could be introduced as the 1st PIM. An evaluation of the effect of crosslinking density and blocking treatment on selective detection indicated that highly selective and sensitive detection of PSA was achieved. Furthermore, the 2nd PIM to introduce fluorescent molecules into the nanocavities led to the fluorescent detection of PSA. The new sequential PIM strategy using multi-functional PIR can potentially create various sophisticated artificial molecular recognition materials.