Enzyme-free sensitive SERS biosensor for the detection of thalassemia-associated microRNA-210 using a cascade dual-signal amplification strategy.

BACKGROUND: ß-thalassemia is a blood disorder caused by autosomal mutations. Gene modulation therapy to activate the γ-globin gene to induce fetal hemoglobin (HbF) synthesis has become a new option for the treatment of ß-thalassemia. MicroRNA-210 (miR-210) contributes to studying the mechanism regulating γ-globin gene expression and is a potential biomarker for rapid ß-thalassemia screening. Traditional miRNA detection methods perform well but necessitate complex and time-consuming miRNA sample processing. Therefore, the development of a sensitive, accurate, and simple miRNA level monitoring method is essential. RESULTS: We have developed a non-enzymatic surface-enhanced Raman scattering (SERS) biosensor utilizing a signal cascade amplification of catalytic hairpin assembly reaction (CHA) and proximity hybridization-induced hybridization chain reaction (HCR). Au@Ag NPs were used as the SERS substrate, and methylene blue (MB)- modified DNA hairpins were used as the SERS tags. The SERS assay involved two stages: implementing the CHA-HCR cascade signal amplification strategy and conducting SERS measurements on the resulting product. The HCR was started by the products of target-triggered CHA, which formed lengthy nicked double-stranded DNA (dsDNA) on the Au@Ag NPs surface to which numerous SERS tags were attached, leading to a significant increase in the SERS signal intensity. High specificity and sensitivity for miR-210 detection was achieved by monitoring MB SERS intensity changes. The suggested SERS biosensor has a low detection limit of 5.13 fM and is capable of detecting miR-210 at concentration between 10 fM and 1.0 nM. SIGNIFICANCE: The biosensor can detect miR-210 levels in the erythrocytes of ß-thalassemia patients, enabling rapid screening for ß-thalassemia and suggesting a novel approach for investigating the regulation mechanism of miR-210 on γ-globin gene expression. In the meantime, this innovative technique has the potential to detect additional miRNAs and to become an important tool for the early diagnosis of diseases and for biomedical research.

Determination of Aminophylline in Human Serum Using Hydrogel Microspheres for Coupled Surface-Enhanced Raman Spectroscopy (SERS) and Solid-Phase Extraction.

Aminophylline (AMP) is a bronchodilator. The therapeutic and toxic doses are very close. Therefore, therapeutic drug monitoring (TDM) of AMP is essential in clinical practice. Microgels were synthesized by free radical precipitation polymerization. Silver@poly(N-isopropyl acrylamide) (Ag@PNIPAM) hybrid microgels were obtained by loading silver (Ag) nanoparticles into the three-dimensional network of the microgels by in situ reduction. The microgel is a three-dimensional reticular structure with tunable pore size, large specific surface area, and good biocompatibility, which can be used as a sorbent for solid-phase extraction (SPE) of target molecules in complex matrices and as a surface-enhanced Raman spectroscopy (SERS) substrate. We optimized the conditions affecting SERS enhancement, such as silver nitrate (AgNO3) concentration and SPE time, according to the SERS strategy of Ag@PNIPAM hybrid microgels to achieve label-free TDM for trace AMP in human serum. The results showed good linearity between the logarithmic concentration of AMP and its SERS intensity in the range of 1-1.1 × 102 µg/mL, with a correlation coefficient (R2) of 0.9947 and a low detection limit of 0.61 µg/mL. The assay accuracy was demonstrated by spiking experiments, with recoveries ranging from 93.0 to 101.8%. The method is rapid, sensitive, reproducible, requires simple sample pretreatment, and has good potential for use in clinical treatment drug monitoring.

Effect of monodisperse mesoporous bioactive glass spheres (MBGs) on the mechanical properties and bioactivity of dental composites.

Secondary caries is one of the main reasons for the failure of dental resin composites, and adding bioactive fillers such as bioactive glass and amorphous calcium phosphate to the resin composites has been proved to be an effective solution for this problem. In the present study, we investigated the effect of monodisperse mesoporous bioactive glass spheres (MBGs) we prepared on the mechanical properties and bioactivity of dental resins. The results revealed that compared with traditional bioactive glass (BG), MBGs fillers significantly enhanced the mechanical properties of the dental resin composites, whether they were added alone or as functional fillers together with nonporous silica particles. The dental resins filled with bimodal fillers (mass ratio of MBGs: nonporous silica = 10:50, total filler loading 60 wt%) exhibited the best mechanical performance. Their flexural strength was 37.66% higher than the samples with BG at the same filling proportion. Furthermore, the prepared MBGs possessed excellent monodispersity and sufficient apatite formation performance, and the biocompatibility of the composites were also improved by MBGs fillers. These suggest the potential use of the prepared MBGs as multifunctional fillers for the improvement of the performance of dental resins.

Preparation of Ca doped wrinkled porous silica (Ca-WPS) for the improvement of apatite formation and mechanical properties of dental resins.

The purpose of this work was to fabricate and characterize Ca doped wrinkled porous silica (Ca-WPS), and evaluate their effect on the mineralization and mechanical properties of resin composites as functional fillers. Ca-WPS were prepared by sol-gel method and characterized by scanning electron microscopy, transmission electron microscopy and N2 adsorption-desorption measurements. The mineralization properties of the prepared Ca-WPS particles and the resin composites with different amount of Ca-WPS were evaluated by simulated body fluid (SBF) immersion method. The mechanical properties (flexural strength, flexural modulus, compressive strength and microhardness) of the dental resins containing unimodal Ca-WPS fillers and bimodal Ca-WPS fillers with nonporous silica were evaluated by a universal testing machine. Results showed that after immersing in SBF for 5 d, apatite formed on the surface of Ca-WPS and composites containing Ca-WPS fillers, indicating the excellent mineralization property of the prepared Ca-WPS. The mechanical properties of the dental resins increase with the increase of the proportion of unimodal Ca-WPS fillers. The dental resins with bimodal Ca-WPS fillers showed better mechanical properties than the group with only nonporous fillers at the same filler loading (60 wt%). Among all the samples, the dental composites filled with bimodal fillers (mass ratio of Ca-WPS: nonporous silica = 10:50, total filler loading 60 wt%) exhibited the best mechanical performance. The flexural strength, flexural modulus, compressive strength and microhardness of these samples were 26.96%, 42.75%, 16.04% and 54.1% higher than the composites with solid silica particles alone, respectively. Thus, the prepared Ca-WPS could effectively improve the apatite formation and mechanical properties of resin composites.

SERS-ELISA determination of human carboxylesterase 1 using metal-organic framework doped with gold nanoparticles as SERS substrate.

By in situ synthesis of gold nanoparticles (AuNPs) within the acid-etched (AE) MIL-101 (Cr) framework, AE-MIL-101 (Cr) nanocomposites embedded with AuNPs (AuNP/AE-MIL-101 (Cr)) were prepared as surface-enhanced Raman scattering (SERS) substrate. AuNPs are uniformly distributed and stabilized inside the metal-organic framework (MOF), thus forming more SERS hotspots. The SERS performance of AuNP/AE-MIL-101 (Cr) was evaluated using 4-mercaptophenylboronic acid (4-MPBA), 4-mercaptobenzoic acid (4-MBA), benzidine, and rhodamine 6G (R6G). The SERS substrate displays satisfying stability with very low background signal. When benzidine is used as the Raman reporter, the limit of detection (LOD) can reach 6.7 × 10-13 mol·L-1, and the relative standard deviation (RSD) of the intra- and inter-batch repetitive tests is less than 5.2%. On this basis, we developed a method for the detection of human carboxylesterase 1 (hCE 1) in human serum using AuNP/AE-MIL-101 (Cr) nanocomposite as SERS substrate and enzyme-linked immunosorbent assay (ELISA) colorimetric substrate as SERS marker. This method was used to determine hCE 1 in clinical serum samples without complicated sample pretreatment, and the detection results were consistent with the data determined by ELISA. In the concentration range 0.1-120 ng·mL-1, the SERS signal intensity of benzidine at 1609 cm-1 gradually decreases with the increase of hCE 1 concentration (R2 = 0.9948). The average recoveries of hCE 1 in human serum are in the range 84 to 108%, with RSDs lower than 7.7%. By using AuNP/acid etching-MIL-101(Cr) metal organic framework (MOF) as SERS substrate and enzyme-linked immunosorbent assay (ELISA) colorimetric substrate as the SERS marker, a rapid and sensitive method for the determination of human carboxylesterase 1 (hCE1) in human serum samples has been developed.

A magnetic SERS immunosensor for highly sensitive and selective detection of human carboxylesterase 1 in human serum samples.

Hepatocellular carcinoma (HCC) is a common and lethal cancer. New serum markers for detecting HCC are urgently needed. Human carboxylesterase 1 (hCE1) is an important member of the serine hydrolase superfamily and is closely related to the occurrence of HCC. It can be used as a good serum marker for early diagnosis of HCC. Here, we developed a surface enhanced Raman scattering (SERS)- based magnetic immunosensor that specifically recognizes and detects trace amounts of hCE1 in human serum via a sandwich structure consisting of a SERS tags, magnetic supporting substrates, and target antigen (hCE1). The SERS tags are 4-mercaptobenzoic acid (4-MBA)-labeled AgNPs, and the SERS supporting substrates are composed of a raspberry-like morphology of Fe3O4@SiO2@AgNPs magnetic nanocomposites surface-functionalized with a hCE1 antibody. The prepared SERS magnetic immunosensor exhibits excellent selectivity and extremely high sensitivity for hCE1 detection. The SERS signal and logarithm of hCE1 concentration presented a wide linear response range of 0.1 ng mL-1 to 1.0 mg mL-1, and the detection limit of hCE1 was 0.1 ng mL-1. The results indicate that the immunosensor can be used for the rapid determination of hCE1 in human serum without a complicated sample pre-treatment. Furthermore, the immunosensor has good reproducibility and stability, and has a promising prospect for the quantitative detection of other tumor markers in early clinical diagnosis.

A boronate-modified molecularly imprinted polymer labeled with a SERS-tag for use in an antibody-free immunoassay for the carcinoembryonic antigen.

An antibody-free immunoassay that makes use of a boronate affinity molecularly imprinted polymer (MIP) and surface enhanced Raman scattering (SERS) tags is described. It was applied to the specific determination of the carcinoembryonic antigen (CEA) in human serum. For the preparation of the boronate affinity array, a polymer capable of adsorbing glycoproteins was first synthesized on the surface of a glass slide with four spots using 4-vinylbenzeneboronic acid (VPBA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the crosslinking agent, and ethylene glycol and cyclohexanol as porogens. The surface of the VPBA-Co-EGDMA can bind target glycoproteins. After specific capture of the glycoprotein, a "MIP-target glycoprotein-SERS tag" sandwich structure was formed by covalent interaction between the SERS nanotag (consisting of gold nanoparticles and 4-mercaptophenylboronic acid [MPBA]). CEA can be quantified in spiked serum with a detection limit of 0.1 ng·mL-1 via the specific Raman band at 1098 cm-1. Graphical abstractSchematic representation of the boronate affinity molecularly imprinted polymer (MIPs) array-based SERS sensor for rapid and sensitive detection of the carcinoembryonic antigen (CEA) from human serum. The boronate affinity MIPs array are used as capture probes, and MPBA@AuNPs are used as SERS tags.