Improving Sensitivity and Reproducibility of Surface-Enhanced Raman Scattering Biochips Utilizing Magnetoplasmonic Nanoparticles and Statistical Methods.

Surface-enhanced Raman scattering (SERS) technology has been widely recognized for its remarkable sensitivity in biochip development. This study presents a novel sandwich immunoassay that synergizes SERS with magnetoplasmonic nanoparticles (MPNs) to improve sensitivity. By taking advantage of the unique magnetism of these nanoparticles, we further enhance the detection sensitivity of SERS biochips through the applied magnetic field. Despite the high sensitivity, practical applications of SERS biochips are often limited by the issues of stability and reproducibility. In this study, we introduced a straightforward statistical method known as "Gaussian binning", which involves initially binning the two-dimensional Raman mapping data and subsequently applying Gaussian fitting. This approach enables a more consistent and reliable interpretation of data by reducing the variability inherent in Raman signal measurements. Based on our method, the biochip, targeting for C-reactive protein (CRP), achieves an impressive detection limit of 5.96 fg/mL, and with the application of a 3700 G magnetic field, it further enhances the detection limit by 5.7 times, reaching 1.05 fg/mL. Furthermore, this highly sensitive and magnetically tunable SERS biochip is easily designed for versatile adaptability, enabling the detection of other proteins. We believe that this innovation holds promise in enhancing the clinical applicability of SERS biochips.

Antiviral therapy reduces hepatocellular carcinoma through suppressing hepatitis B virus replication may improve ER stress, mitochondrial and metabolic dysfunctions and decrease p62 in hybridized mice with single HBV transgene and miR-122.

Hepatitis B virus (HBV) hijacks autophagy for its replication. Nucleos(t)ide analogs (NUCs) treatment suppressed HBV replication and reduced hepatocellular carcinoma (HCC) incidence. However, the use of NUCs in chronic hepatitis B (CHB) patients with normal or minimally elevated serum alanine aminotransferase (ALT) levels is still debated. Animal models are crucial for studying the unanswered issue and evaluating new therapies. MicroRNA-122 (miR-122), which regulates fatty acid and cholesterol metabolism, is downregulated during hepatitis and HCC progression. The reciprocal inhibition of miR-122 with HBV highlights its role in HCC development as a tumor suppressor. By crossbreeding HBV-transgenic mice with miR-122 knockout mice, we generated a hybrid mouse model with a high incidence of HCC up to 89% and normal ALT levels before HCC. The model exhibited early-onset hepatic steatosis, progressive liver fibrosis, and impaired late-phase autophagy. Metabolomics and microarray analysis identified metabolic signatures, including dysregulation of lipid metabolism, inflammation, genomic instability, the Warburg effect, reduced TCA cycle flux, energy deficiency, and impaired free radical scavenging. Antiviral treatment reduced HCC incidence in hybrid mice by approximately 30-35% compared to untreated mice. This effect was linked to the activation of ER stress-responsive transcription factor ATF4, clearance of autophagosome cargo p62, and suppression of the CHOP-mediated apoptosis pathway. In summary, this study suggests that despite minimal ALT elevation, HBV replication can lead to liver injury. Endoplasmic reticulum stress, reduced miR-122 levels, mitochondrial and metabolic dysfunctions, blocking protective autophagy resulting in p62 accumulation, apoptosis, fibrosis, and HCC. Antiviral may improve the above-mentioned pathogenesis through HBV suppression.

A simple and fast method for the fabrication of p-typeß-Ga2O3by electrochemical oxidation method with DFT interpretation.

In this work, a simple electrochemical oxidation method has been used to prepare p-typeß-Ga2O3nanoparticles. This method overcomes the problem of doping high energy gap semiconductors to form p-type. The electron holes ofß-Ga2O3were caused by oxygen vacancy (Vo) and showed the shorter lattice constant and preferred orientation in XRD analysis. The peak area of oxygen vacancy also reflects a higher ratio than n-type Ga2O3in x-ray photoelectron spectroscopy (XPS). The adsorption of reducing gas (CO, CH4, and H2) enhanced the resistance of theß-Ga2O3confirming the p-type character of NPs. The DFT calculations showed that oxygen vacancy leads to higher energy of the Fermi level and is near the valence band. The binding energy of Ga2O3and after interaction with gas molecular was also calculated which is analogous to our experimental data.

Sensitivity enhancement of magneto-optical Faraday effect immunoassay method based on biofunctionalized γ-Fe2O3@Au core-shell magneto-plasmonic nanoparticles for the blood detection of Alzheimer's disease.

In this work, we conducted a proof-of-concept experiment based on biofunctionalized magneto-plasmonic nanoparticles (MPNs) and magneto-optical Faraday effect for in vitro Alzheimer's disease (AD) assay. The biofunctionalized γ-Fe2O3@Au MPNs of which the surfaces are modified with the antibody of Tau protein (anti-τ). As anti-τ reacts with Tau protein, biofunctionalized MPNs aggregate to form magnetic clusters which will hence induce the change of the reagent's Faraday rotation angle. The result showed that the γ-Fe2O3@Au core-shell MPNs can enhance the Faraday rotation with respect to the raw γ-Fe2O3 nanoparticles. Because of their magneto-optical enhancement effect, biofunctionalized γ-Fe2O3@Au MPNs effectively improve the detection sensitivity. The detection limit of Tau protein as low as 9 pg/mL (9 ppt) was achieved. Furthermore, the measurements of the clinical samples from AD patients agreed with the CDR evaluated by the neurologist. The results suggest that our method has the potential for disease assay applications.

A magneto-optical biochip for rapid assay based on the Cotton-Mouton effect of γ-Fe2O3@Au core/shell nanoparticles.

BACKGROUND: In the past decades, different diseases and viruses, such as Ebola, MERS and COVID-19, impacted the human society and caused huge cost in different fields. With the increasing threat from the new or unknown diseases, the demand of rapid and sensitive assay method is more and more urgent. RESULTS: In this work, we developed a magneto-optical biochip based on the Cotton-Mouton effect of γ-Fe2O3@Au core/shell magnetic nanoparticles. We performed a proof-of-concept experiment for the detection of the spike glycoprotein S of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The assay was achieved by measuring the magneto-optical Cotton-Mouton effect of the biochip. This magneto-optical biochip can not only be used to detect SARS-CoV-2 but also can be easily modified for other diseases assay. CONCLUSION: The assay process is simple and the whole testing time takes only 50 min including 3 min for the CM rotation measurement. The detection limit of our method for the spike glycoprotein S of SARS-CoV-2 is estimated as low as 0.27 ng/mL (3.4 pM).

Improvement of multisource localization of magnetic particles in an animal.

In this simulation work, the linearized Bregman iterative algorithm was applied to solve the magnetic source distribution problem of a magnetic particle imaging (MPI) system for small animals. MPI system can apply an excitation magnetic field, and the induced magnetic field from the magnetic nanoparticles (MNPs) can be detected by the sensors of MPI system. With a gaussian distribution source at the upper side of the mouse brain, sensors set above the mouse brain and the constant excitation magnetic field, the average deviation of the calculated source distribution from the multiplane scanning along the axis away from the mouse brain and the closest plane scanning are 2.78 × 10-3 and 2.84 × 10-3 respectively. The simulated result showed that combination of multiplane scanning hardly improves the accuracy of the source localization. In addition, a gradient scan method was developed that uses gradient magnetic field to scan the mouse brain. The position of the maximum of the lead field matrix will be controlled by the gradient field. With a set up gaussian distribution source at the bottom of the mouse brain, the average deviation of the calculated source distribution from the gradient scan method and the constant field are 4.42 × 10-2 and 5.05 × 10-2. The location error from the two method are 2.24 × 10-1 cm and 3.61 × 10-1 cm. The simulation showed that this method can improve the accuracy compared to constant field when the source is away from the sensor and having a potential for application.

Magneto-Optical Characteristics of Streptavidin-Coated Fe3O4@Au Core-Shell Nanoparticles for Potential Applications on Biomedical Assays.

Recently, gold-coated magnetic nanoparticles have drawn the interest of researchers due to their unique magneto-plasmonic characteristics. Previous research has found that the magneto-optical Faraday effect of gold-coated magnetic nanoparticles can be effectively enhanced because of the surface plasmon resonance of the gold shell. Furthermore, gold-coated magnetic nanoparticles are ideal for biomedical applications because of their high stability and biocompatibility. In this work, we synthesized Fe3O4@Au core-shell nanoparticles and coated streptavidin (STA) on the surface. Streptavidin is a protein which can selectively bind to biotin with a strong affinity. STA is widely used in biotechnology research including enzyme-linked immunosorbent assay (ELISA), time-resolved immunofluorescence (TRFIA), biosensors, and targeted pharmaceuticals. The Faraday magneto-optical characteristics of the biofunctionalized Fe3O4@Au nanoparticles were measured and studied. We showed that the streptavidin-coated Fe3O4@Au nanoparticles still possessed the enhanced magneto-optical Faraday effect. As a result, the possibility of using biofunctionalized Fe3O4@Au nanoparticles for magneto-optical biomedical assays should be explored.

Crystal Structure of a Homogeneous IgG-Fc Glycoform with the N-Glycan Designed to Maximize the Antibody Dependent Cellular Cytotoxicity.

N-glycosylation on IgG modulates Fc conformation and effector functions. An IgG-Fc contains a human sialo-complex type (hSCT) glycan of biantennary structure with two α2,6-sialylations and without core-fucosylation is an optimized glycoform developed to enhance the antibody dependent cellular cytotoxicity (ADCC). hSCT modification not only enhances the binding affinity to Fc receptors in the presence of antigen but also in some cases provides gain-of-function effector activity. We used enzymatic glyco-engineering to prepare an IgG-Fc with homogeneous hSCT attached to each CH2 domain and solved its crystal structure. A compact form and an open form were observed in an asymmetric unit in the crystal. In the compact structure, the double glycan latches from the two hSCT chains stabilize the CH2 domains in a closed conformation. In the open structure, the terminal sialic acid (N-acetylneuraminic acid or NeuNAc) residue interacts through water-mediated hydrogen bonds with the D249-L251 helix, to modulate the pivot region of the CH2-CH3 interface. The double glycan latches and the sialic acid modulation may be mutually exclusive. This is the first crystal structure of glyco-engineered Fc with enhanced effector activities. This work provides insights into the relationship between the structural stability and effector functions affected by hSCT modification and the development of better antibodies for therapeutic applications.

An Effective Bacterial Fucosidase for Glycoprotein Remodeling.

Fucose is an important component of many oligo- and polysaccharide structures as well as glycoproteins and glycolipids, which are often associated with a variety of physiological processes ranging from fertilization, embryogenesis, signal transduction, and disease progression, such as rheumatoid arthritis, inflammation, and cancer. The enzyme α-l-fucosidase is involved in the cleavage of the fucosidic bond in glycans and glycoconjugates, particularly the Fuc-α-1,2-Gal, Fuc-α-1,3/4-GlcNAc, and Fuc-α-1,6-GlcNAc linkages. Here, we report a highly efficient fucosidase, designated as BfFucH identified from a library of bacterial glycosidases expressed in E. coli from the CAZy database, which is capable of hydrolyzing the aforementioned fucosidic linkages, especially the α-1,6-linkage from the N-linked Fuc-α-1,6-GlcNAc residue on glycoproteins. Using BfFucH coupled with endoglycosidases and the emerging glycosynthases allows glycoengineering of IgG antibodies to provide homogeneous glycoforms with well-defined glycan structures and optimal effector functions.

A common glycan structure on immunoglobulin G for enhancement of effector functions.

Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.

Glycan array on aluminum oxide-coated glass slides through phosphonate chemistry.

A new type of glycan array covalently or noncovalently attached to aluminum oxide-coated glass (ACG) slides has been developed for studies of enzymatic reactions and protein binding. To prepare the noncovalent array, glycans with a polyfluorinated hydrocarbon (-C(8)F(17)) tail are spotted robotically onto the ACG slide surface containing a layer of polyfluorinated hydrocarbon terminated with phosphonate. After incubation and washing, the noncovalent array can be characterized by MS-TOF via ionization/desorption at a low laser energy without addition of matrix. A representative cellotetraose array was developed to study the activity and specificity of different cellulases and to differentiate the exo- and endoglucanase activities. To prepare the covalent array, glycans with a phosphonic acid tail were synthesized and spotted robotically onto the ACG slide surface. After incubation, the slides can be used directly for quantitative protein binding analysis. Compared to the preparation of glycan arrays on glass slides and other surfaces, this method of arraying using phosphonic acid reacting with ACG is more direct, convenient, and effective and represents a new platform for the high-throughput analysis of protein-glycan interactions.

Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer.

Cancer-associated carbohydrate antigens are often found on the surface of cancer cells. Understanding their roles in cancer progression will lead to the development of new therapeutics and high-sensitivity diagnostics for cancers. Globo H is a member of this family, which is highly expressed on breast cancer cells. Here, we report the development of a glycan microarray of Globo H and its analogs for measurement of the dissociation constants on surface (K(D,surf)) with three different monoclonal antibodies (VK-9, Mbr1, and anti-SSEA-3), to deduce their binding specificity. The glycan microarray was also used to detect the amount of antibodies present in the plasma of breast cancer patients and normal blood donors. It was shown that the amount of antibodies against Globo H from breast cancer patients were significantly higher than normal blood donors, providing a new tool for possible breast cancer diagnosis. Compared with the traditional ELISA method, this array method required only atto-mole amounts of materials and is more effective and more sensitive (5 orders of magnitude). The glycan microarray thus provides a new platform for use to monitor the immune response to carbohydrate epitopes after vaccine therapy or during the course of cancer progression.

Glycan arrays on aluminum-coated glass slides.

We have developed a novel method of immobilizing glycans onto aluminum-coated glass (ACG) slides for potential use in disease diagnosis and drug discovery. The quality of these sugar chips can be assessed by mass spectrometry and fluorescence measurements with high sensitivity. The unique properties of ACG slides include: 1) the metal oxide layer on the surface can be activated for grafting organic compounds such as modified oligosaccharides; 2) the surface remains electrically conductive, and the grafted oligosaccharides can be simultaneously characterized by mass spectrometry and carbohydrate-binding assay; and 3) the slides are more sensitive than transparent glass slides in binding analysis. To demonstrate this, we synthesized a model compound of mannose with a built-in photocleavable linker bound to the ACG slide surface. The molecular weight of the grafted mannose was identified by mass spectrometry, and the slide was subjected to biotinylated ConA binding followed by Cy3-tagged streptavidin detection. This method was further extended to the preparation of glycan arrays containing lactose and the cancer antigen Globo H.

Homodinuclear iron thiolate nitrosyl compounds [(ON)Fe(S,S-C6H4)2 Fe(NO)2]- and [(ON)Fe(SO2,S-C6H4)(S,S-C6H4)Fe(NO)2]- with {Fe(NO)}7-{Fe(NO)2}9 electronic coupling: new members of a class of dinitrosyl iron complexes.

Reaction of Fe(CO)2(NO)2 and [(ON)Fe(S,S-C6H3R)2]- (R = H (1), CH3 (1-Me))/[(ON)Fe(SO2,S-C6H4)(S,S-C6H4)]- (4) in THF afforded the diiron thiolate/sulfinate nitrosyl complexes [(ON)Fe(S,S-C6H3R)2 Fe(NO)2]- (R = H (2), CH3 (2-Me)) and [(ON)Fe(S,SO2-C6H4)(S,S-C6H4)Fe(NO)2]- (3), respectively. The average N-O bond lengths ([Fe(NO)2] unit) of 1.167(3) and 1.162(4) A in complexes 2 and 3 are consistent with the average N-O bond length of 1.165 A observed in the other structurally characterized dinitrosyl iron complexes with an {Fe(NO)2}9 core. The lower nu(15NO) value (1682 cm(-1) (KBr)) of the [(15NO)FeS4] fragment of [(15NO)Fe(S,S-C6H3CH3)2 Fe(NO)2]- (2-Me-15N), compared to that of [(15NO)Fe(S,S-C6H3CH3)2]- (1-Me-15N) (1727 cm(-1) (KBr)), implicates the electron transfer from {Fe(NO)2}10 Fe(CO)2(NO)2 to complex 1-Me/1 may occur in the process of formation of complex 2-Me/2. Then, the electronic structures of the [(NO)FeS4] and [S2Fe(NO)2] cores of complexes 2, 2-Me, and 3 were best assigned according to the Feltham-Enemark notation as the {Fe(NO)}7-{Fe(NO)2}9 coupling (antiferromagnetic interaction with a J value of -182 cm(-1) for complex 2) to account for the absence of paramagnetism (SQUID) and the EPR signal. On the basis of Fe-N(O) and N-O bond distances, the dinitrosyliron {L2Fe(NO)2} derivatives having an Fe-N(O) distance of approximately 1.670 A and a N-O distance of approximately 1.165 A are best assigned as {Fe(NO)2}9 electronic structures, whereas the Fe-N(O) distance of approximately 1.650 A and N-O distance of approximately 1.190 A probably imply an {Fe(NO)2}10 electronic structure.

Methane emission from fields with three various rice straw treatments in Taiwan paddy soils.

Flooded rice fields are one of the major biogenic methane sources. In this study, the effects of straw residual treatments on methane emission from paddy fields were discussed. The experimental field was located at Tainan District Agricultural Improvement Station in Chia-Yi county (23 degrees 25'08''N, 120degrees16'26''E) of southern Taiwan throughout the first and the second crop seasons in 2000. The seasonal methane fluxes in the first crop season with rice stubble removed, rice straw burned and rice straw incorporated were 4.41, 3.78 and 5.27 g CH4 m(-2), and the values were 32.8, 38.9 and 75.1 g CH4 m(-2) in the second crop season, respectively. In comparison of three management methods of rice straw residue, the incorporation of rice straw residue should show a significant tendency for enhancing methane emission in the second crop season. Moreover, stubble removed and straw burned treatments significantly reduced CH4 emissions by 28 approximately 56% emissions compared to straw incorporated plot. Concerning for air quality had led to legislation restricting rice straw burning, removing of rice stubble might be an appropriate methane mitigation strategy in Taiwan paddy soils.

Methane emission from fields with differences in nitrogen fertilizers and rice varieties in Taiwan paddy soils.

Flooded rice fields are one of the major biogenic methane sources. In this study, methane emission rates were measured after transplanting in paddy fields with application of two kinds of nitrogen fertilizers (ammonium sulfate, NH4+-N and potassium nitrate, NO3(-)-N) and with two kinds of rice varieties (Japonica and Indica). The experiment was conducted in fields located at Tainan District Agricultural Improvement Station in Chia-Yi county (23 degrees 25'08"N, 120 degrees 16'26"E) of southern Taiwan throughout the first and the second crop seasons in 1999. The seasonal methane flux in the first crop season with NH4+-N and NO3(-)-N ranged from 2.48 to 2.78 and from 8.65 to 9.22 g CH4 m(-2); and the values ranged 24.6-34.2 and 36.4-52.6 g CH4 m(-2) in the second crop season, respectively. In the first crop season, there were significantly increased 3.1-3.7-fold in methane emission fluxes due to plantation of Indica rice. In comparison of two rice varieties, the Indica rice variety showed a tendency for larger methane emission than the Japonica rice variety in the second crop season. Moreover, ammonium sulfate treatment significantly reduced CH4 emissions by 37-85% emissions compared to potassium nitrate plots. It was concluded that the CH4 emission was markedly dependent on the type of nitrogen fertilizer and rice variety in Taiwan paddy soils.