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.

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.

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.

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.