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.

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.

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.

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.

Molecularly Imprinted Polymer Arrays as Synthetic Protein Chips Prepared by Transcription-type Molecular Imprinting by Use of Protein-Immobilized Dots as Stamps.

Molecularly imprinted polymer (MIP) arrays were demonstrated for the recognition of proteins. They were prepared via transcription-type molecular imprinting where patterned dots composed of biotinylated nanoparticles were first immobilized on a glass substrate followed by the immobilization of versatile biotinylated proteins via avidin-biotin interactions, yielding a multiple protein-immobilized stamp as a mold that could be transcribed. MIPs were prepared between the stamp and a methacrylated glass substrate, and after the stamp was peeled off, MIP dots were able to be prepared on the methacrylated glass substrate according to the positions of the immobilized proteins on the stamp. We confirmed that the prepared MIP array showed the expected selective binding toward the corresponding template proteins by conducting competitive binding assays using the fluorescently labeled proteins as corresponding competitors. The binding behaviors were consistent with those obtained by a surface plasmon resonance sensing system. We believe that the proposed platform involving the easily handled nanoparticle-based protein stamps for the preparation of MIP arrays can provide a new type of pattern recognition-based protein chip, which can be adopted as a substitute for the use of conventional protein arrays in various research and industrial fields in the life sciences.

Synthesis of Monodispersed Submillimeter-Sized Molecularly Imprinted Particles Selective for Human Serum Albumin Using Inverse Suspension Polymerization in Water-in-Oil Emulsion Prepared Using Microfluidics.

We synthesized monodispersed submillimeter-sized (100 µm-1 mm) microgels by inverse suspension polymerization of water-soluble monomer species with a photoinitiator in water-in-oil (W/O) droplets formed by the microchannel. After fundamental investigations of the selection of suitable surfactants, surfactant concentration, and flow rate, we successfully prepared monodispersed submillimeter-sized W/O droplets. Because radical polymerization based on thermal initiation was not appropriated based on colloidal stability, we selected photoinitiation, which resulted in the successful synthesis of monodispersed submillimeter-sized microgels with sufficient colloidal stability. The microgel size was controlled by the flow rate of the oil phase, which maintained the monodispersity. In addition, the submillimeter-sized microgels exhibit high affinity and selective binding toward HSA utilizing molecular imprinting. We believe the monodispersed submillimeter-sized molecularly imprinted microgels can be used as affinity column packing materials without any biomolecules, such as antibodies, for sample pretreatment to remove unwanted proteins without a pump system.

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.

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.

SPR sensing of bisphenol A using molecularly imprinted nanoparticles immobilized on slab optical waveguide with consecutive parallel Au and Ag deposition bands coexistent with bisphenol A-immobilized Au nanoparticles.

A slab-type optical waveguide (s_OWG)-based microfluidic SPR measurement system for bisphenol A was developed. This s_OWG possesses consecutive parallel gold and silver deposition bands in the line of plasmon flow, allowing two individual SPR signals to be independently obtained as a result of the difference in resonant reflection spectra of these metals. As a molecular recognition element, molecularly imprinted polymer nanoparticles (MIP-Np) were employed and immobilized on the surface of each of the gold and silver deposition bands. The resonant reflection spectra were measured on the MIP-Np-immobilized consecutive parallel gold and silver deposition bands coexistent with BPA-AuNp. The Ag-based SPR spectra showed a red shift (0.7 nm) when free BPA (0.1 mM) was passed over the BPA-AuNp/immobilized MIP-Np complexes formed on the s_OWG, unlike the case for the Au deposition band, while a large excess of BPA induced a blue shift due to the competitive desorption of BPA-AuNp from the immobilized MIP-Np on the s_OWG. By using the proposed detection system, binding events of other small molecules could be monitored in conjunction with the use of MIP-Np and labeled-AuNp.

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.

[An anesthetic case of a patient with multiple sclerosis and supranuclear palsy].

A 60-year-old female with multiple sclerosis (MS) and supranuclear palsy (PSP) was scheduled for right eye iridotomy, left eye phaco emulcification aspiration and insertion of the intraocular lens. Her medical conditions included prolonged immobility, spastic contracture, and a history of convulsion. She was administered with L-dopa, tizanidine, bacrofen, and dantrorane. Anesthesia was induced with propofol 50 mg and fentanyl 25 microg intravenously, and inhalation of oxygen and 1% sevoflurane. Tracheal intubation was performed without neuromuscular blocking agents. Anesthesia was maintained with inhalation of oxygen-air (Fi(O2) 0.4) and 1-1.5% sevoflurane, combined with regional anesthesia. Supplemental fentanyl was administered as needed. The bispectral index (BIS) was monitored and kept between 40 and 60. The operation proceeded uneventfully. After discontinuation of anesthetic agents, she awoke immediately and the BIS index returned to the pre-induction level. Neither neurological disturbances nor unexpected event were observed postoperatively. In patients with MS, it is important to remember the possibility of drugs moving into the central nervou system easily due to the disturbance of the blood-brai barrier. Patients with PSP are usually medicated wit. various medicines which have possibility of interactin with anesthetics. Therefore, we used least anesthetic as possible. In this case, monitoring of BIS seemed to be useful to maintain the minimum sevoflurane concen trations needed.

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.

Continuous Repetitive Data Acquisition with 123I-FP-CIT SPECT: Effects of Rotation Speed and Rotation Time.

The aim of this study was to evaluate the effects of the acquisition rotation speed and the rotation time for continuous repetitive rotation acquisition (CRRA) on image quality and quantification in 123I-FP-CIT SPECT. Methods: An anthropomorphic striatal phantom filled with 123I solution was acquired with CRRA and the step-and-shoot (SS) mode. The following combinations of acquisition rotation speed and rotation time for CRRA were used: 0.50 rpm by 30 frames, 0.17 rpm by 10 frames, 0.10 rpm by 6 frames, and 0.05 rpm by 3 frames. SPECT images were reconstructed using ordered-subset expectation maximization with resolution recovery, scatter, and CT-based attenuation correction. Two kinds of image processing patterns-image reconstruction after the addition of projection data (the added-projection-data process) and image addition after data reconstruction (the added-reconstructed-image process)-were investigated in this study. The effects of the acquisition parameters and the image processes were evaluated by the full width at half maximum, percentage coefficient of variation (%CV), and specific binding ratio (SBR). Results: With full width at half maximum, there were no clear differences between CRRA images obtained with the various rotation speeds before rotation and the SS mode. Although the combination of a slow rotation speed and a short rotation time improved image uniformity compared with the SS mode, the %CV obtained by CRRA increased as the rotation speed increased. The %CVs were 11.9% ± 0.9% for 0.50 rpm by 30 frames, 6.9% ± 0.9% for 0.05 rpm by 3 frames, and 9.6% ± 0.5% for SS mode. SBRs obtained by CRRA with the added-projection-data process were equal to those obtained by SS mode. However, SBRs obtained with the added-reconstructed-image process were clearly decreased compared with the SS mode. Conclusion: The combination of rotation speed and rotation times affects the image quality and quantification of 123I-FP-CIT SPECT using CRRA. When CRRA is applied in 123I-FP-CIT SPECT, it is necessary to use added-projection-data processes and proper rotation speeds (e.g., 0.10-0.17 rpm rotation speed).

A molecularly imprinted nanocavity-based fluorescence polarization assay platform for cortisol sensing.

We prepared core-shell-type molecularly imprinted polymer particles (MIP-NPs) for cortisol using cortisol-21-monomethacrylate as a template molecule, itaconic acid as an additional functional monomer, styrene as a comonomer and divinylbenzene as a crosslinker, and established a fluorescence polarization-based sensing nano-platform for the competitive binding assay of cortisol using dansyl-labeled cortisol (dansyl-cortisol). Before the preparation of MIP-NPs, the binding behavior of bulk MIPs prepared by conventional radical polymerization was preliminarily characterized. NIPs prepared with methacrylic acid instead of cortisol-21-monomethacrylate showed less binding activity than the MIPs, revealing that the molecular imprinting process enhanced the affinity toward cortisol. Since the imprinting effect was confirmed in this system, the fluorescence polarization-based sensing nano-platform for cortisol was constructed using MIP-NPs with dansyl-cortisol, where the binding event of cortisol was transduced into the fluorescence anisotropy change of dansyl-cortisol from the bound-state to the free-state, on the basis of the concentration-dependent competitive replacement of dansyl-cortisol by cortisol added on MIP-NPs. The complex of MIP-NPs with dansyl-cortisol was more effectively formed than that of the reference polymer particles (R-MIP-NPs) prepared without itaconic acid, suggesting that the itaconic acid and cortisol-21-monomethacrylate-derived methacrylic acid residues can work cooperatively. Highly sensitive cortisol detection was achieved by the proposed molecularly imprinted nanocavity-based fluorescence polarization assay for cortisol sensing with dansyl-cortisol, and the apparent limit of detection was estimated to be ca. 80 nM.

Post-imprinting and In-Cavity Functionalization.

Molecularly imprinted polymers (MIPs) are artificial materials capable of molecular recognition for target molecules. Currently MIPs have been prepared without further modification after polymerization, and used for predetermined single purposes. Post-imprinting modifications (PIMs) presented here can provide site-specific modifications within the molecularly imprinted binding cavities after polymerization, enabling MIPs to become more complex functional materials as were the cases of naturally occurring conjugated proteins. We present an overview of the research on MIPs involving PIMs, including transformation of binding sites, on/off switching of binding activity, introduction of desirable functions such as fluorescent signalling functions, catalytic activity, and so on. The combination of PIMs with molecular imprinting appears to be a powerful tool for preparing a diverse range of biomimetic functional materials.

Molecularly imprinted microspheres for bisphenol a prepared using a microfluidic device.

Spherical molecularly imprinted polymer particles for bisphenol A (BPA-MIP) were easily prepared by using a Y-junction microfluidic device. The sizes of the obtained BPA-MIP particles were found to be 86 µm with a narrow size distribution. The binding characteristics were investigated by drawing a binding isotherm to estimate the binding constant and by switching the polarity of solvents to examine the feasibility of use as a medium for affinity chromatography. When dichloromethane was used as a solvent, BPA was strongly bound to the spherical BPA-MIP particles based on hydrogen bond formation; after switching the solvent to methanol, BPA was eluted quantitatively due to the weakening of the hydrogen bonding, suggesting that the spherical BPA-MIP particles can be applied to affinity-type solid-phase extraction for BPA. As the present method can provide a diverse range of spherical MIPs without tedious procedures, MIP-based affinity media will be able to be more readily used as pretreatment and/or purification for various fields.