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To apply semiconductor nanostructures as a solid matrix for laser desorption/ionization-mass spectrometry, the nanostructures should exhibit ionization activity through photocatalytic reactions and desorption enhancement through photothermal conversion. Herein, TiO2 aerogels were synthesized using titanium-(IV) butoxide as a precursor, and a controlled porous structure was obtained by adjusting the ethanol content and annealing temperatures. The photocatalytic activity of the TiO2 aerogel was estimated using pseudo-first-order kinetics on methylene blue degradation reactions. To achieve further desorption/ionization properties, a combi-matrix was produced using the TiO2 aerogel and a conventional organic matrix, α-cyano-4-hydroxynnamic acid (CHCA). The enhanced ionization efficiency of the TiO2 aerogel combi-matrix was demonstrated along with an improved signal-to-noise ratio from other solid matrix combi-matrix systems. Meanwhile, the distribution of CHCA crystals in TiO2 aerogel with various annealing conditions was investigated by differential scanning calorimetry with the Gibbs–Thomson equation. The radius of CHCA crystals was minimized at 550 °C but increased steeply at heat treatment temperatures higher than 600 °C, implying a collapse in the pore structure of the TiO2 aerogel. Furthermore, the activation energy for melting the CHCA nanocrystals was calculated using the Arrhenius equation. These results showed that CHCA nanocrystals in a combi-matrix with the TiO2 aerogel after annealing at 550 °C could be most effectively melted with lower activation energy via the pore structure and the binary phased TiO2 aerogel. Finally, the combi-matrix based on TiO2 aerogel was applied for the analysis of the sera from sepsis patients (n = 40), systemic inflammatory response syndrome (SIRS) patients (n = 20), and healthy controls (n = 20), finding that the combi-matrix based on TiO2 aerogel could be effectively used for the diagnosis of sepsis using lysophosphatidylcholine (LPC-16) as a biomarker. [ABSTRACT FROM AUTHOR]

The SiO2 aerogel has a porous network of spherical silica particles with an average size of 3–5 nm, which provides an extremely high surface area. Herein, SiO2 aerogel was synthesized using the sol–gel method with sodium silicate as a precursor, and the SiO2 aerogel was applied to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as an inorganic matrix for quantitative analysis. In this work, a combi-matrix was prepared by mixing the SiO2 aerogel and a conventional organic matrix (CHCA) for quantitative analysis of analytes at an m/z ratio of less than 500. Using the combi-matrix based on the SiO2 aerogel with an extremely high porosity, analyte ionization was performed without the mass peaks of the organic matrix (CHCA) for MALDI-MS. The optimal ratio between the SiO2 aerogel and the organic matrix was determined to eliminate the mass peaks from the organic matrix and maximize the signal-to-noise ratio (S/N ratio) of the analytes. Quantitative analysis was performed using the optimal combi-matrix system (a mixture of SiO2 aerogel and organic matrix (CHCA)). The homogeneous and dense distribution of analytes was demonstrated using fluorescence imaging, and the spot-to-spot and shot-to-shot reproducibility was estimated using the SiO2 aerogel. Finally, LDI-MS based on the combi-matrix was applied for the quantitative analysis of a biomarker for colon cancer, dodecanoyl-l-carnitine (DC). Using real samples from healthy volunteers and patients, a medical diagnosis of colon cancer was demonstrated using a combi-matrix. [ABSTRACT FROM AUTHOR]

Inhibitors of the epithermal growth factor receptor (EGFR) were screened from an autodisplayed Fv-antibody library using an anti-EGF antibody. The Fv-antibody library was expressed on the outer membrane of Escherichia coli , which corresponds to the heavy chain V H region of immunoglobulin G. The library was constructed by randomizing the CDR3 region of expressed V H regions (11 amino acid residues) by site-directed mutagenesis. Using an anti-EGF antibody as a screening probe, amino acid sequences (CDR3 region) with antibody binding affinity were screened from the Fv-antibody library. These amino acid sequences were considered to have similar chemical properties to EGF, which can bind to EGFR. Two autodisplayed clones with Fv-antibodies against EGFR were screened from the Fv-antibody library, and the screened Fv-antibodies were expressed as soluble proteins. The binding affinity ( K D ) was estimated using an SPR biosensor, and the inhibitory activity of expressed Fv-antibodies was observed for PANC-1 pancreatic tumor cells and T98G glioblastoma cells using Western blot analysis of proteins in the EGFR-mediated signaling pathway. The viability of PANC-1 and T98G cells was observed to decrease via the inhibitory activity of expressed Fv-antibodies. Finally, interactions between Fv-antibodies and EGFR were analyzed by using molecular docking simulations.

Serotonin-like mimotopes were screened from the Fv-antibody library to be used as inhibitors against monoamine oxidase A (MAO-A). The Fv-antibody [corresponding to the V H region of immunoglobulin G (IgG)] consists of three complementarity-determining regions and four frame regions. The Fv-antibody library was prepared by site-directed mutagenesis of CDR3, which consists of 11 amino acid residues. Three target clones were screened from the Fv-antibody library, and the binding affinity of the screened clones to the monoclonal anti-serotonin antibody was analyzed using fluorescence-activated cell sorting. The screened Fv-antibodies were expressed as soluble proteins fused with green fluorescence protein. Additionally, the screened CDR3 regions (11 residues) of the selected Fv-antibodies were synthesized as peptides with linking amino acid residues. The binding constants ( K D ) of the three serotonin-like mimotopes (Fv-antibodies and peptides) were estimated using a surface plasmon resonance biosensor. The inhibitory activity (IC 50 ) of the serotonin-like mimotopes (Fv-antibodies and peptides) was estimated separately for MAO-A and MAO-B enzymes and compared with that of conventional inhibitors. Finally, the screened serotonin-like mimotopes were used to treat a cell line (SH-SY5Y, ATCC code: CRL-2266) expressing serotonin receptors. This was done to confirm the following two aspects: (1) the binding of mimotopes to the serotonin receptors on the cell surface and (2) the inhibitory activity of mimotopes against MAO-A enzymes in the cell lysates., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

A defect-passivated photosensor based on cesium lead bromide (CsPbBr 3 ) perovskite quantum dots (QD) was fabricated using parylene films, and the photosensor was applied for the microbial detection. The CsPbBr 3 perovskite QDs were synthesized to be homogeneous in size under thermodynamic control, and the perovskite QD-based photosensor was fabricated using MoS 2 flakes as the electron transfer layer. In this work, a parylene film with functional groups was deposited on a photosensor for physical protection (waterproof) and defect (halide vacancy) passivation of the perovskite QD. As the first effect of the parylene film, the physical protection of the perovskite QD from water was estimated by comparing the photosensor performance after incubation in water. As the second effect of the parylene, the interaction between the functional groups of the parylene film and the halide vacancies of the perovskite QDs was investigated through the bandgap, crystal structure, and trap-state density analysis. Additionally, density functional theory analysis on Mulliken charges, lattice parameters, and Gibbs free energy demonstrated the effect of the defect passivation by parylene films. Finally, the parylene-passivated QD-based photosensor was applied to the detection of two kinds of food-poisoning and gastroduodenal disease bacteria ( Listeria monocytogenes and Helicobacter pylori ).

Pancreatic ribonuclease A (RNase A) inhibitors were screened from an autodisplayed Fv-antibody library, which was prepared by randomizing amino acid sequences of the third complementary-determining region (CDR3) within the heavy chain variable region (V H region) of immunoglobulin G (called "Fv-antibody" comprising three CDRs and four frame regions (FRs)) through site-directed mutagenesis. The library was autodisplayed on the outer membrane of Escherichia coli . Target Fv-variants (clones) with specific binding affinity for RNase A were screened using fluorescein-labeled RNase A and flow cytometry. Three Fv variants (clones) were screened, and CDR3 amino acid sequences were analyzed. The screened Fv-antibodies were expressed as soluble proteins, and CDR3 was synthesized into peptides (11 residues). The binding affinity constants ( K D ) of the expressed Fv-antibodies and synthesized peptides to RNase A were estimated using surface plasmon resonance. Fitting analysis based on the adsorption model showed that K D values of the three expressed Fv-antibodies were estimated to be 17.5 ± 4.1, 28.8 ± 9.7, and 33.9 ± 8.9 nM ( n = 3), and those of the three synthesized peptides were 1.3 ± 0.1, 1.3 ± 0.3, and 3.7 ± 1.3 μM ( n = 3). From the RNase activity assay with an RNA probe labeled with fluorophore and quencher, inhibition constants (IC 50 ) of the three expressed Fv-antibodies were estimated to be 90.2, 65.3, and 98.8 nM ( n = 3), and those of the three synthesized peptides were 8.1, 3.6, and 0.4 μM ( n = 3). The activity of RNase inhibitors constituting the expressed Fv-antibodies and synthesized peptides was demonstrated via an RNA cleavage test using the total RNA from HeLa cells., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Inhibitors for monoamine oxidase-B (MAO-B) were screened from an F V library with a randomized complementarity-determining region 3 (CDR3) region using a monoclonal antibody against dopamine. As the first step, the F V library was expressed on the outer membrane of E. coli by site-directed mutagenesis of the randomized CDR3 region. Among the F V library, variants with a binding affinity to monoclonal antibodies against dopamine were screened and cloned. From the comparison of the binding activity of the screened clones to a control clone with a modified F V antibody (only with CDR1 and CDR2), the CDR3 regions of screened clones were determined to directly interact with the monoclonal antibody against dopamine. These CDR3 sequences were then synthesized as mimotopes (mimicking peptides) of dopamine. The inhibitory activity of two mimotopes against MAO-B was analyzed using HeLa cells overexpressing MAO-B, as well as using activated human astrocytes; their inhibitory activity was compared to that of a commercial inhibitor of MAO-B, selegiline. The inhibition efficiency of the two mimotopes (in comparison with selegiline) was estimated to be 67.2% and 69.4% in the HeLa cells and 64.4% and 58.0% in the human astrocytes. The gene expression pattern in astrocytes after treatment with the two mimotopes was also analyzed and compared with that in the human astrocytes treated with selegiline. Finally, the interaction between two mimotopes and MAO-B was analyzed using docking simulation, and the candidate regions of MAO-B for the interaction with each mimotope were explored through the docking simulation.

Switching peptides were designed to bind reversibly to the binding pocket of antibodies (IgG) by interacting with frame regions (FRs). These peptides can be quantitatively released when antigens bind to IgG. As FRs have conserved amino acid sequences, switching peptides can be used as antibodies for different antigens and different source animals. In this study, an electrochemical one-step immunoassay was conducted using switching peptides labeled with ferrocene for the quantitative measurement of analytes. For the effective amperometry of the switching peptides labeled with ferrocene, a pyrolyzed carbon electrode was prepared by pyrolysis of the parylene-C film. The feasibility of the pyrolyzed carbon electrode for the electrochemical one-step immunoassay was determined by analyzing its electrochemical properties, such as its low double-layer capacitance ( C dl ), high electron transfer rate ( k app ), and wide electrochemical window. In addition, the factors influencing the amperometry of switching peptides labeled with ferrocene were analyzed according to the hydrodynamic radius, the number of intrahydrogen bonds, dipole moments, and diffusion coefficients. Finally, the applicability of the electrochemical one-step immunoassay for the medical diagnosis of the human hepatitis B surface antigen (hHBsAg) was assessed.

Chemiluminescence immunoassays have been widely employed for diagnosing various diseases. However, because of the extremely low intensity chemiluminescence signals, highly sensitive transducers, such as photomultiplier tubes and image sensors with cooling devices, are required to overcome this drawback. In this study, a hypersensitive photosensor was developed based on cesium lead bromide (CsPbBr 3 ) perovskite quantum dots (QDs) with sufficient high sensitivity for chemiluminescence immunoassays. First, CsPbBr 3 QDs with a highly uniform size, that is, 5 nm, were synthesized under thermodynamic control to achieve a high size confinement effect. For the fabrication of the photosensor, MoS 2 nanoflakes were used as an electron transfer layer and heat-treated at an optimum temperature. Additionally, a parylene-C film was used as a passivation layer to improve the physical stability and sensitivity of the photosensor. In particular, the trap states on the CsPbBr 3 QDs were reduced by the passivation layer, and the sensitivity was increased. Finally, a photosensor based on CsPbBr 3 QDs was employed in chemiluminescence immunoassays for the detection of human hepatitis B surface antigen, human immunodeficiency virus antibody, and alpha-fetoprotein (AFP, a cancer biomarker). When compared with the conventionally used equipment, the photosensor was determined to be feasible for application in chemiluminescence immunoassays.

Crystals of monosodium urate monohydrate (MSU) and calcium pyrophosphate dihydrate (CPPD) are known to induce arthropathic diseases called gout and pseudogout, respectively. These crystals are deposited in various joints or tissues, causing severe pain. Correct identification of crystals is crucial for the appropriate treatment of gout and pseudogout, which exhibit very similar symptoms. Herein, a novel approach of laser desorption/ionization time-of-flight (LDI-ToF) mass spectrometry (MS) was introduced to analyze MSU and CPPD crystals with three different types of nanostructured TiO 2 materials including TiO 2 nanoparticles (P25), TiO 2 nanowires synthesized from wet-corrosion method, and the mixture of P25 and TiO 2 nanowires (P25/TiO 2 nanowires) as inorganic solid matrices. Furthermore, the feasibility of LDI-ToF MS based on these TiO 2 nanostructures for the analysis of the two arthropathy-related crystals was tested using spiked samples in synovial fluid at known crystal concentrations. The mass analysis results of MSU and CPPD crystals demonstrated that (1) the electrostatic interaction between analytes and solid matrices was key for the analyte ionization and (2) LDI-ToF MS with nanostructured TiO 2 materials has the potential to be a practical approach for the diagnosis of gout and pseudogout.

A combination nanostructured matrix with metal Au nanoislands and semiconductor TiO 2 nanowires is presented to enhance both desorption and ionization efficiency in laser desorption/ionization (LDI) mass spectrometry. The heterostructure of Au nanoislands on TiO 2 nanowires was fabricated via (1) TiO 2 nanowire synthesis through a modified wet-corrosion method and (2) Au nanoisland formation through thermal annealing of a sputtered Au layer on the TiO 2 nanowires. Herein, the synergistic effect of this heterostructure for highly efficient ion production was experimentally elucidated in terms of the formation of high temperature on the surface of Au and the creation of a Schottky barrier at the Au-TiO 2 interface. Finally, four types of immunosuppressors were analyzed to demonstrate the improved ionization performance of the heterostructure for LDI mass spectrometry.

In this work, TiO 2 nanowires synthesized from a wet-corrosion process were presented for peptide sequencing by photocatalytic reaction with UV radiation. For the photocatalytic decomposition of peptides, the peptide sample was dropped on a target plate containing synthesized TiO 2 nanowire zones and UV-irradiated. Subsequently, the target plate was analyzed by laser desorption/ionization time-of-flight (LDI-TOF) mass spectrometry using the synthesized TiO 2 nanowires as a solid matrix. The feasibility of peptide sequencing based on the photocatalytic reaction with the synthesized TiO 2 nanowires was demonstrated using six types of peptides GHP9 (G 1 -H-P-Q-G 2 -K 1 -K 2 -K 3 -K 4 , 1006.59 Da), BPA-1(K 1 -S 1 -L-E-N-S 2 -Y-G 1 -G 2 -G 3 -K 2 -K 3 -K 4 , 1394.74 Da), PreS1(F 1 -G-A-N 1 -S-N 2 -N 3 -P 1 -D 1 -W-D 2 -F 2 -N 4 -P 2 -N 5 , 1707.68 Da), HPQ peptide-1 (G-Y-H-P-Q-R-K, 884.45 Da), HPQ peptide-2 (K-R-H-P-Q-Y-G, 884.45 Da), and HPQ peptide-3 (R-Y-H-P-Q-G-K, 884.45 Da). The identification of three different peptides with the same molecular weight was also demonstrated by using the synthesized TiO 2 nanowires for their photocatalytic decomposition as well as for LDI-TOF mass spectrometry as a solid-matrix.

In this work, the chronoamperometry-based redox cycling of 3,3',5,5'-tetramethylbenzidine (TMB) was performed by using interdigitated electrode (IDE). The signal was obtained from two sequential chronoamperometric profiles: (1) with the generator at the oxidative potential of TMB and the collector at the reductive potential of TMB, and (2) with the generator at the reductive potential of TMB and the collector at the oxidative potential of TMB. The chronoamperometry-based redox cycling (dual mode) showed a sensitivity of 1.49 μA/OD, and the redox cycling efficiency was estimated to be 94% (n = 10). The sensitivities of conventional redox cycling with the same interdigitated electrode and chronoamperometry using a single working electrode (single mode) were estimated to be 0.67 μA/OD and 0.18 μA/OD, respectively. These results showed that the chronoamperometry-based redox cycling (dual mode) could be more effectively used to quantify the oxidized TMB than other amperometric methods. The chronoamperometry-based redox cycling (dual mode) was applied to immunoassays using a commercial ELISA kit for medical diagnosis of the human hepatitis B virus surface antigen (hHBsAg). Finally, the chronoamperometry-based redox cycling (dual mode) provided more than a 10-fold higher sensitivity than conventional chronoamperometry using a single working electrode (single mode) when applied to a commercial ELISA kit for medical diagnosis of hHBsAg.

Commercial ELISA kits for substance P (SubP), which are helpful for the clinical diagnosis of acute myocardial infarction, are limited in efficacy because of low sensitivity. A highly sensitive immunoassay was developed using silica spheres encapsulating a quantum dot-layer (SQS) and labeling antibodies, on a Parylene A-modified plate. The high sensitivity was possible by taking advantage of the enhanced photoluminescence of the SQS and dense immobilization of SubP on a Parylene A-modified plate. Glutaraldehyde was used for cross-linking of SQS to the anti-SubP antibody and SubP to the Parylene A coating. The SQS-linked immunosorbent assay (SQSLISA) was optimized and validated. The dynamic range for the assay was 1-10000 pg/mL with a linear correlation factor of 0.9992 when the competitive SQSLISA was employed. The intra- and interday accuracies were 93-100% and 87-122%, respectively. The reproducibility was lower than 11%. The developed method was applied to clinical samples collected from healthy controls (n = 30) and acute myocardial infarction (n = 16) and it displayed a high correlation with the commercial ELISA kit, with a limit of detection that was 30-fold lower. Clinical sample analysis confirmed that SubP is a promising diagnostic marker for acute myocardial infarction. The SQSLISA is expected to be a practical and useful assay tool.