Investigation of lignin substructures participating in self-assembly for the synthesis of monodisperse lignin spherical particles.

The intricate structure of lignin, characterized by a mix of hydrophilic components and hydrophobic structures from its aliphatic and aromatic constituents, poses challenges in creating monodisperse particles. This is due to the need for precise modulation of self-assembly kinetics. Herein, we explore a correlation between the substructure of lignin and its capacity for self-assembly. We have conducted an in-depth investigation into the interactions between hydrophilic groups, such as phenolic and aromatic-OH, and monolignols with interunit linkages that are involved in the formation of lignin particles (LPs). A high degree of hydrophilicity with a condensed structure is crucial for high supersaturation levels, which in turn determines the growth phase and leads to small LPs. An approach based on tailoring the supersaturation level which is contingent on the structural characteristics of extracted organosolv lignin was used to obtain remarkably uniform LPs with mean diameters of approximately 230 and 480 nm. The results of this study have the potential to serve as a foundation for the preparation of monodisperse LPs derived from various lignin sources as well as for the development of methods to extract lignin containing a specific chemical substructure.

ß-Ketoadipic acid production from poly(ethylene terephthalate) waste via chemobiological upcycling.

The upcycling of poly(ethylene terephthalate) (PET) waste can simultaneously produce value-added chemicals and reduce the growing environmental impact of plastic waste. In this study, we designed a chemobiological system to convert terephthalic acid (TPA), an aromatic monomer of PET, to ß-ketoadipic acid (ßKA), a C6 keto-diacid that functions as a building block for nylon-6,6 analogs. Using microwave-assisted hydrolysis in a neutral aqueous system, PET was converted to TPA with Amberlyst-15, a conventional catalyst with high conversion efficiency and reusability. The bioconversion process of TPA into ßKA used a recombinant Escherichia coli ßKA expressing two conversion modules for TPA degradation (tphAabc and tphB) and ßKA synthesis (aroY, catABC, and pcaD). To improve bioconversion, the formation of acetic acid, a deleterious factor for TPA conversion in flask cultivation, was efficiently regulated by deleting the poxB gene along with operating the bioreactor to supply oxygen. By applying two-stage fermentation consisting of the growth phase in pH 7 followed by the production phase in pH 5.5, a total of 13.61 mM ßKA was successfully produced with 96% conversion efficiency. This efficient chemobiological PET upcycling system provides a promising approach for the circular economy to acquire various chemicals from PET waste.

Modulation of the peroxidase-like activity of iron oxide nanoparticles by surface functionalization with polysaccharides and its application for the detection of glutathione.

Here, we employed three polysaccharides, such as dextran, hyaluronic acid, and chitosan, for surface modification of iron oxide nanoparticles (IONPs) and carried out in-depth investigation to elucidate the effect of surface functionalities on the peroxidase (POD) like activity of IONPs. The affinity of substrates to the catalytic site of IONPs was found to be determined by the surface functional groups and hydration layer of polysaccharide coating on the surface of IONPs. The role of hydration layer was further confirmed by the results that the POD-like activity of IONPs coated with a certain polysaccharide having higher water holding capacity was significantly enhanced by salting-out reagent, such as ammonium chloride that is known to reduce the thickness of hydration layer. Moreover, the excellent catalytic activity of dextran-coated IONPs was successfully applied to develop a highly sensitive sensing system for the detection of glutathione (GSH) with a limit of detection of 2.3 nM.

Synthetic Ligand-Coated Starch Magnetic Microbeads for Selective Extraction of Food Additive Silicon Dioxide from Commercial Processed Food.

The amorphous form of silicon dioxide has long been regarded as a safe food additive (E551) that is widely used in commercially processed food as an anticaking agent. However, starting with titanium dioxide, there have been growing safety concerns regarding to the use of nanoscale silicon dioxide particles in food as food additives. The size, morphology, and chemical properties of inorganic food materials are important parameters to determine its potential toxicity. Therefore, an effective means of extracting an intact form of SiO2 from food without altering the physicochemical property of SiO2 particles is of great need to accurately monitor its characteristics. Here, we report on an effective magnetic separation method to extract food additive SiO2 from food by utilizing a diatom-originated peptide with a specific affinity to SiO2 particles. The affinity-based magnetic separation was found to be specific to SiO2 particles over other types of inorganic food additives such as titanium dioxide and zinc oxide. The size and morphology of SiO2 were shown to not be affected by the extraction processes. This method was successfully applied to extract and characterize the food additive SiO2 from six different types of commercial food.

Paper-based colorimetric detection of pathogenic bacteria in food through magnetic separation and enzyme-mediated signal amplification on paper disc.

Herein, we report a colorimetric sensing system for the detection of highly virulent bacteria, Escherichiacoli O157:H7, in sausage by utilizing magnetic separation and enzyme-mediated signal amplification on paper disc. For magnetic separation, Poly-l-lysine coated starch magnetic particles (PLL@SMPs) were synthesized and utilized for the separation and concentration of the bacteria in sample suspension. Horseradish peroxidase-conjugated antibody (HRP-Antibody) and 3,3',5,5'- tetramethylbenzidine (TMB) were employed for the specific signal amplification in the presence of target bacteria. The synthesized PLL@SMPs showed an excellent capture efficiency (>90%) for the pathogenic bacteria in large volume sample suspension. The intrinsic problems associated with the non-specific binding of sensing components that lead to the high background signal and low sensitivity in colorimetric detection was successfully resolved by employing hyaluronic acid as a blocking agent. The effective separation and concentration of target bacteria by PLL@SMPs and target-specific signal amplification with exceptionally high signal to noise ratio enabled the detection of target bacteria with a detection limit in the single digit regime. The sensing system proposed in this study was successfully used for the detection of the target pathogenic bacteria, E. coli O157:H7, in sausage sample with the limit of detection (LOD) as low as 30.8 CFU/mL with 95% probability. The simple nature of paper-based detection system with a great sensitivity and specificity would provide an effective means of evaluating the safety of food and environmental samples.

Topological analysis of single-stranded DNA with an alpha-hederin nanopore.

Nanopores have been emerged as a powerful tool for analyzing the structural information and interactional properties of a range of biomolecules. The spatial resolution of nanopore is determined by the diameter and effective thickness of its constriction region, but the presence of vestibule or stem structure in protein-based nanopore could negatively affect the sensitivity of the nanopore when applied for genome sequencing and topological analysis of DNA. Recently, alpha-hederin (Ah) has been reported to form a sub-nanometer scale pore structure in lipid membrane. With the simple structure and extremely small effective thickness, the Ah nanopore was shown to discriminate four different types of nucleotides. However, identification of a certain nucleotide in a strand of DNA, which is essential for genome sequencing, remains challenging. Here, we investigated the resolving capability of Ah nanopore to discriminate few nucleotides in a strand of single-stranded DNA, and the factors determining the sensitivity of Ah nanopore. The Ah nanopore was shown to be able to identify as few as three adenosine nucleotides in a strand of poly cytidine, in which the dwell time of the additional current blockade that represents the adenosine residue was in good agreement with their physical length. We also found that the lateral tension and chain pressure generated around the nanopore were influenced by pore's diameter and played as a dependent variables to determine the geometry of nanopore's constriction as well as the spatial resolution of the Ah nanopore.

Gold Nanoparticle-Coated Starch Magnetic Beads for the Separation, Concentration, and SERS-Based Detection of E. coli O157:H7.

Here, we report gold nanoparticle-coated starch magnetic beads (AuNP@SMBs) that were prepared by in situ synthesis of AuNPs on the surface of SMBs. Upon functionalization of the surface with a specific antibody, the immuno-AuNP@SMBs were found to be effective in separating and concentrating the target pathogenic bacteria, Escherichia coli O157:H7, from an aqueous sample as well as providing a hotspot for surface-enhanced Raman scattering (SERS)-based detection. We employed a bifunctional linker protein, 4× gold-binding peptide-tagged Streptococcal protein G (4GS), to immobilize antibodies on AuNP@SMBs and AuNPs in an oriented form. The linker protein also served as a Raman reporter, exhibiting a strong and unique fingerprint signal during the SERS measurement. The amplitude of the SERS signal was shown to have a good correlation with the concentration of target bacteria ranging from 100 to 105 CFU/mL. The detection limit was determined to be as low as a single cell, and the background signals derived from nontarget bacteria were negligible due to the excellent specificity and colloidal stability of the immuno-AuNP@SMBs and SERS tags. The highly sensitive nature of the SERS-based detection system will provide a promising means to detect the pathogenic microorganisms in food or clinical specimen.

Paper-Based Radial Chromatographic Immunoassay for the Detection of Pathogenic Bacteria in Milk.

Here, a paper-based radial flow chromatographic immunoassay (RFCI) employing gold nanoparticles (AuNPs) as chromatic agents was developed for the detection of Escherichia coli O157:H7 in whole milk. A 4-repeated gold-binding peptide-tagged (4GBP) streptococcal protein G (SPG) fusion protein was constructed as a bifunctional linker to immobilize antibodies on the surface of AuNPs with a well-oriented form based on the specific affinity of GBP and SPG to the gold and Fc portion of the antibody, respectively. 4GS@AuNPs prepared with the bifunctional linker protein exhibited excellent colloidal stability even at high salt concentrations of up to 500 mM, which is a critical requirement for its application to a broad range of biological and food samples. The enhanced colloidal stability and excellent binding capability of the immuno-4GS@AuNPs toward target bacteria lowered the detection limit of RFCI for target pathogenic bacteria in whole milk as low as 103 CFU/mL, which is by an order of magnitude lower than that of conventional immuno-AuNPs prepared with physical adsorption of antibodies. The RFCI pattern could also be converted into a grayscale value by simple image processing for quantitative determination of target pathogenic bacteria. This paper-based detection system would provide an effective means of monitoring the presence of food-borne pathogens in real food samples with naked eyes.

Colorimetric Determination of the Activity of Starch-Debranching Enzyme via Modified Tollens' Reaction.

Nelson-Somogyi and 3,5-dinitrosalicylic acid (DNS) assays are the classical analytical methods for the determination of activity of starch-debranching enzymes, however, they have a narrow detection range and do not adapt to the quantitative measurement of linear polysaccharides. Herein, we developed a simple and accurate colorimetric assay for determining the activity of starch-debranching pullulanase through the modified Tollens' reaction in combination with UV irradiation. Silver nanoparticles (AgNPs) were formed by reducing aldehyde groups in short-chain glucans (SCGs) generated by debranching of waxy maize starch using pullulanase through the modified Tollens' reaction. In addition to providing a reducing moiety to the Tollens' reaction, the debranching product, SCGs, also enhanced the colloidal stability of synthesized AgNPs, of which the amplitude of its surface plasmon resonance (SPR) absorbance peak was proportional to the concentration of SCGs ranging from 0.01-10 mg/mL. The detection limit of this system was 0.01 mg/mL, which was found to be 100 times higher than that of the conventional DNS assay. The purification of SCGs by recrystallization and gelatinization improved the selectivity of this colorimetric assay for debranching products, which provides a simple and accurate means of monitoring the debranching process and characterizing the activity of starch-debranching enzymes.

Molecular Rearrangement of Glucans from Natural Starch To Form Size-Controlled Functional Magnetic Polymer Beads.

Herein, we report a fairly simple and environmentally friendly approach for the fabrication of starch-based magnetic polymer beads (SMPBs) with uniform shape and size through spontaneous rearrangement of short-chain glucan (SCG) produced by enzymatic debranching of waxy maize starch. The paramagnetic materials, dextran-coated iron oxide nanoparticles (Dex@IONPs), were readily incorporated into the starch microstructure and rendered a superparamagnetic property to the SMPBs. The morphology and size of resulting SMPBs turned out to be modulated by Dex@IONPs in a concentration-dependent manner, of which Dex@IONPs was assumed to be acting as a seed inducing the epitaxial crystallization of SCG and further transforming it into homogeneous microparticles. The surface of SMPBs was readily functionalized with an antibody through a one-step reaction using a linker protein. The immuno-SMPBs showed great capture efficiency (>90%) for target bacteria. The colloidal stability and favorable surface environment for biomolecules are believed to be responsible for the high capture efficiency and specificity of the SMPBs. Furthermore, the captured bacteria along with antibody and linker protein were effectively eluted from the surface of SMPBs by adding free maltose, making this new material suitable for various chromatographic applications.