Epidermal Growth Factor Receptor (EGFR) Inhibitors Screened from Autodisplayed Fv-Antibody Library.

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 VH region of immunoglobulin G. The library was constructed by randomizing the CDR3 region of expressed VH 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 (KD) 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.

Comparative In Vitro Dissolution Assessment of Calcined and Uncalcined Hydroxyapatite Using Differences in Bioresorbability and Biomineralization.

This study reports the effect of the not-calcining process on the bioresorption and biomineralization of hydroxyapatite through in vitro dissolution assessment. The prepared calcined hydroxyapatite (c-HAp) and uncalcined hydroxyapatite (unc-HAp) have a particle size of 2 µm and 13 µm, surface areas of 4.47 m2/g and 108.08 m2/g, and a Ca/P ratio of 1.66 and 1.52, respectively. In vitro dissolution assessments of c-HAp and unc-HAp were performed for 20 days at 37 °C in a citric acid buffer according to ISO 10993-14. During the dissolution, the c-HAp and unc-HAp confirmed an increase in weight, and the calcium and phosphorous ions were rapidly released. The calcium ions released from c-HAp formed rod-shaped particles with a longer and thinner morphology, while in unc-HAp, they appeared thicker and shorter. In the ICP-OES results, the concentrations of calcium elements were initially increased and then decreased by this formation. The rod-shaped particles identified as calcium citrate (Ca-citrate) through the XRD pattern. The calcium content of Ca-citrate particles from unc-HAp was higher than that from c-HAp. The unc-HAp demonstrated non-toxic properties in a cytotoxicity evaluation. Therefore, due to its higher bioresorption and biomineralization, unc-HAp exhibits enhanced biocompatibility compared to c-HAp.

Breathing-Driven Self-Powered Pyroelectric ZnO Integrated Face Mask for Bioprotection.

Rapid spread of infectious diseases is a global threat and has an adverse impact on human health, livelihood, and economic stability, as manifested in the ongoing coronavirus disease 2019 (COVID-19) pandemic. Even though people wear a face mask as protective equipment, direct disinfection of the pathogens is barely feasible, which thereby urges the development of biocidal agents. Meanwhile, repetitive respiration generates temperature variation wherein the heat is regrettably wasted. Herein, a biocidal ZnO nanorod-modified paper (ZNR-paper) composite that is 1) integrated on a face mask, 2) harvests waste breathing-driven thermal energy, 3) facilitates the pyrocatalytic production of reactive oxygen species (ROS), and ultimately 4) exhibits antibacterial and antiviral performance is proposed. Furthermore, in situ generated compressive/tensile strain of the composite by being attached to a curved mask is investigated for high pyroelectricity. The anisotropic ZNR distortion in the bent composite is verified with changes in ZnO bond lengths and OZnO bond angles in a ZnO4 tetrahedron, resulting in an increased polarization state and possibly contributing to the following pyroelectricity. The enhanced pyroelectric behavior is demonstrated by efficient ROS production and notable bioprotection. This study exploring the pre-strain effect on the pyroelectricity of ZNR-paper might provide new insights into the piezo-/pyroelectric material-based applications.

A vertically paired electrode for redox cycling and its application to immunoassays.

An electrochemical immunoassay based on the redox cycling method was presented using vertically paired electrodes (VPEs), which were fabricated using poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) as an electrode material and parylene-C as a dielectric layer. For the application to immunoassays, different electrochemical properties of PEDOT:PSS were analyzed for the redox reaction of 3,3',5,5'-tetramethylbenzidine (TMB, the chromogenic substrate for enzyme-immunoassays) at different pH conditions, including the conductivity (σ), electron transfer rate constant (kapp), and double-layer capacitance (Cdl). The influencing factors on the sensitivity of redox cycling based on VPE based on PEDOT:PSS were analyzed for the redox reaction of TMB, such as the electrode gap and number of electrode pairs. Computer simulation was also performed for the redox cycling results based on VPEs, which had limitations in fabrication, such as VPEs with an electrode gap of less than 100 nm and more than five electrode pairs. Finally, the redox cycling based on VPE was applied to the medical diagnosis of human hepatitis-C virus (hHCV) using a commercial ELISA kit. The sensitivity of the redox cycling method for the medical diagnosis of hHCV was compared with conventional assay methods, such as TMB-based chromogenic detection, luminol-based chemiluminescence assay, and a rapid test kit (lateral flow immunoassay).

Effects of hole transporting PEDOT:PSS on the photoemission of upconverted hot electron in Mn-doped CdS/ZnS quantum dots.

In this work, we investigated the effect of hole transporting poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) interfacing with Mn-doped CdS/ZnS quantum dots (QDs) deposited on an indium tin oxide (ITO) substrate on the photoemission of upconverted hot electrons under weak continuous wave photoexcitation in a vacuum. Among the various factors that can influence the photoemission of the upconverted hot electrons, we studied the role of PEDOT:PSS in facilitating the hole transfer from QDs and altering the energy of photoemitted hot electrons. Compared to hot electrons emitted from QDs deposited directly on the ITO substrate, the addition of the PEDOT:PSS layer between the QD and ITO layers increased the energy of the photoemitted hot electrons. The increased energy of the photoemitted hot electrons is attributed in part to the reduced steady-state positive charge on the QDs under continuous photoexcitation, which reduces the energy required to eject the electron from the conduction band.

Homogeneous One-Step Immunoassay Based on Switching Peptides for Detection of the Influenza Virus.

In this study, a homogeneous one-step immunoassay based on switching peptides is presented for the detection of influenza viruses A and B (Inf-A and Inf-B, respectively). The one-step immunoassay represents an immunoassay method that does not involve any washing steps, only treatment of the sample. In this method, fluorescence-labeled switching peptides quantitatively dissociate from the antigen-binding site of immunoglobulin G (IgG). In particular, the one-step immunoassay based on soluble detection antibodies with switching peptides is called a homogeneous one-step immunoassay. The immunoassay developed uses switching peptides labeled with two types of fluorescence dyes (FAM and TAMRA) and detection antibodies labeled with two types of fluorescence quenchers (TQ2 for FAM and TQ3 for TAMRA). The optimal switching peptides for the detection of Inf-A and Inf-B have been selected as L1-peptide and H2-peptide. The interactions between the four kinds of switching peptides and IgG have been analyzed using computational docking simulation and SPR biosensor. The location of labeling for the fluorescence quenchers has been determined based on the distance between the fluorescence dyes of the switching peptides and the fluorescence quenchers, calculated on the basis of the efficiency of fluorescence quenching, using the Förster equation. To demonstrate the feasibility of the one-step immunoassay, binding constants (KD) have been calculated for detection antibodies against Inf-A and Inf-B with target antigens (Inf-A and Inf-B) and switching peptides (L1- and H2-peptides), using an isotherm model. The immunoassay has been demonstrated to be feasible using antigens as well as real samples of Inf-A and Inf-B with a critical cycle number (Ct). The immunoassay has also been compared to other commercially available rapid test kits for Inf-A and Inf-B and found to be far more sensitive for detection of Inf-A and Inf-B over the entire detection range.

Antibody-Mediated Screening of Peptide Inhibitors for Monoamine Oxidase-B (MAO-B) from an Autodisplayed FV Library.

Inhibitors for monoamine oxidase-B (MAO-B) were screened from an FV library with a randomized complementarity-determining region 3 (CDR3) region using a monoclonal antibody against dopamine. As the first step, the FV library was expressed on the outer membrane of E. coli by site-directed mutagenesis of the randomized CDR3 region. Among the FV 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 FV 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.

Overexpression of CYP11A1 recovers cell cycle distribution in renal cell carcinoma Caki-1.

BACKGROUND: Clear cell renal carcinoma is commonly known for its metastasis propensity to outspread to other organs and is asymptomatic in the early stage. Recent studies have shown that deficiencies in CYP11A1 expression can lead to fatal adrenal failure if left untreated and are associated with downstream regulation in various cancer types. However, the molecular mechanisms of CYP11A1 and kidney cancer proliferation remain unclear. METHODS: Normal and renal carcinoma cell lines (HEK293 and Caki-1) were transfected with plasmid encoding CYP11A1 to overexpress the P450scc protein. Cell cycle distribution was investigated using flow cytometry. The expression of proteins related to C-Raf/ERK/JNK/p38 signaling pathways was examined using western blot. RESULTS: We observed that CYP11A1 overexpression suppressed the cyclin B1 and cell-division cycle 2 expression while cyclin-dependent kinases 2 and 4 were unaffected. Cancer cell migration and invasion were suppressed along with epithelial-intermediate metastatic markers Snail and Vimentin. In addition, in CYP11A1-overexpressing Caki-1 cells, cdc2/cyclinB1 was downregulated while the phosphorylation of cdc25c, a G2/M arrest-related upstream signal, was increased. The intrinsic-mitochondrial apoptosis markers were not significantly altered. We also identified that the C-Raf/ERK/JNK/p38 pathway is an important pro-apoptotic mechanism in CYP11A1-overexpressing cell-based models. Our results suggest that CYP11A1 overexpression recovered the disturbed cell cycle arrest distribution in renal carcinoma cell line Caki-1 through G2/M arrest and C-Raf/ERK/JNK pathway. CONCLUSIONS: Our findings may suggest promising new therapeutic targets to suppress kidney cancer proliferation without affecting normal cells, eventually improving the survival of patients with cancer.

Simultaneous analysis of acylcarnitines using MALDI-TOF mass spectrometry based on a parylene matrix chip.

Short and medium chain acylcarnitines have been used for the diagnosis of various fatty acid oxidation and organic acid disorders. This report presents a multiplex and quantitative analysis of acylcarnitines using MALDI-TOF MS based on a parylene matrix chip. The parylene matrix chip was fabricated by the deposition of a nanoporous film of parylene on an organic matrix array, which reduced the number of mass peaks from the organic matrix in the low m/z range. Quantitative analysis was possible using the parylene matrix chip because of the formation of nano-sized sample crystals on the nanoporous parylene film. Seven acylcarnitines were quantitatively analyzed using the chip; the method detection range included the cut-off values for metabolic disorders. The seven acylcarnitines of different concentrations were simultaneously detected using the parylene matrix chip and the interference from the mixed carnitines was estimated. Real L-carnitine (C0) samples were analyzed using serial dilution, and the recoveries were calculated by comparisons with a standard curve.

Carbon electrode obtained via pyrolysis of plasma-deposited parylene-C for electrochemical immunoassays.

In this study, parylene-C films from plasma deposition as well as thermal deposition were pyrolyzed to prepare a carbon electrode for application in electrochemical immunoassays. Plasma deposition could prepare parylene-C in a faster deposition rate and more precise control over the thickness in comparison with the conventional thermal deposition. To analyze the influence of the deposition method, the crystal and electronic structures of the pyrolyzed parylene-C films obtained via both deposition methods were compared using Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy. For application as a carbon electrode in immunoassays, the electrochemical properties of the pyrolyzed carbon films from two both deposition methods were analyzed, including the double layer capacitance (2.10 µF cm-2 for plasma deposition and 2.20 µF cm-2 for thermal deposition), the apparent electron transfer rate (approximately 1.1 × 10-3 cm s-1 for both methods), and the electrochemical window (approximately -1.0 ∼ 2.1 V for both methods). Finally, the applicability of the pyrolyzed carbon electrode from parylene-C was demonstrated for the diagnosis of human hepatitis-C using various amperometric methods, such as cyclic voltammetry, chronoamperometry, square-wave voltammetry and differential pulse voltammetry.

One-step immunoassay for the detection of food-poisoning related bacteria using a switching peptide.

A one-step immunoassay was developed for five types of food-poisoning-related bacteria using a switching peptide and antibodies isolated from unimmunized horse serum. The one-step immunoassay involves mixing samples and reagents in a homogeneous solution without any washing steps. In this work, a one-step immunoassay configuration was developed using isolated antibodies labelled with an organic fluorescence quencher and a switching-peptide labelled with a fluorescent dye. The fluorescence-labelled switching-peptide was bound to the antigen-binding site of the isolated antibodies before binding to the bacteria (no fluorescence signal), and the switching-peptide dissociated from the antibodies as soon as they bound to the bacteria (fluorescence signal turns on). By quantifying the generated fluorescence signal, the one-step immunoassay presented here allows microbial detection without any washing step.

Photothermal Structural Dynamics of Au Nanofurnace for In Situ Enhancement in Desorption and Ionization.

Fundamental properties of nanostructured substrates govern the performance of laser desorption/ionization mass spectrometry (LDI-MS); however, limited studies have elucidated the desorption/ionization mechanism based on the physicochemical properties of substrates. Herein, the enhancement in desorption/ionization is investigated using a hybrid matrix of Au nanoisland-functionalized ZnO nanotubes (AuNI-ZNTs). The underlying origin is explored in terms of the photo-electronic and -thermal properties of the matrix. This is the first study to report the effect of laser-induced surface restructuring/melting phenomenon on the LDI-MS performance. AuNI plays a central role as a photothermal nanofurnace, which facilitates the internal energy transfer from the AuNI to the adsorbed analytes by reconstruction in the structurally dynamic AuNI and therefore favors the desorption process. Moreover, piezoelectricity is driven in situ in the AuNI-ZNT hybrid, which modulates the overall band structure and thereby promotes the ionization process. Ultimately, high LDI-MS performance is demonstrated by analyzing small metabolites of fatty acids and monosaccharides, which are challenged to be detected in conventional LDI-MS. This study emphasizing the understanding of matrix properties can provide insights into the design and development of a novel nanomaterial as an efficient LDI matrix. Furthermore, the developed hybrid matrix can overcome the major hurdles existing in conventional LDI-MS.

Fluorescein and Rhodamine B-Binding Domains from Autodisplayed Fv-Antibody Library.

In this study, the binding domains for fluorescent dyes were presented that could be used as synthetic peptides or fusion proteins. Fv-antibodies against two fluorescent dyes (fluorescein and rhodamine B) were screened from the Fv-antibody library, which was prepared on the outer membrane of Escherichia coli using the autodisplay technology. Two clones with binding activities to each fluorescent dye were screened separately from the library using flow cytometry. The binding activity of the screened Fv-antibodies on the outer membrane was analyzed using fluorescent imaging with the corresponding fluorescent dyes. The CDR3 regions of the screened Fv-antibodies (11 amino acid residues) were synthesized into peptides, and each peptide was analyzed for its binding activity to each fluorescent dye using fluorescence resonance energy transfer (FRET) experiments. These CDR3 regions were demonstrated to have a binding activity to each fluorescent dye when the regions were co-expressed as a fusion protein with Z-domain.

Modulation of SIRT3 expression through CDK4/6 enhances the anti-cancer effect of sorafenib in hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths worldwide. The only drug currently approved for clinical use in the treatment of advanced HCC is sorafenib. However, many patients with HCC show reduced sensitivity to sorafenib during treatment. SIRT3, a member of the mammalian sirtuin family, is a tumor suppressor in certain tumor types. However, only few studies have investigated the effects of SIRT3 on tumor prognosis and sorafenib sensitivity in patients with HCC. Here, we aimed to investigate the correlation between SIRT3 expression and glucose metabolism and proliferation in HCC and discover effective compounds that increase endogenous SIRT3 modulation effect of sorafenib. METHODS: To determine the correlation between SIRT3 and glucose related proteins, immunostaining was performed with liver cancer tissue using various antibodies. To investigate whether the expression of SIRT3 in HCC is related to the resistance to sorafenib, we treated sorafenib after the modulation of SIRT3 levels in HCC cell lines (overexpression in Huh7, knockdown in HepG2). We also employed PD0332991 to modulate the SIRT3 expression in HCC cell and conducted functional assays. RESULTS: SIRT3 expression was downregulated in high glycolytic and proliferative HCC cells of human patients, xenograft model and HCC cell lines. Moreover, SIRT3 expression was downregulated after sorafenib treatment, resulting in reduced drug sensitivity in HCC cell lines. To enhance the anti-tumor effect of sorafenib, we employed PD0332991 (CDK4/6-Rb inhibitor) based on the correlation between SIRT3 and phosphorylated retinoblastoma protein in HCC. Notably, combined treatment with sorafenib and PD0332991 showed an enhancement of the anti-tumor effect in HCC cells. CONCLUSIONS: Our data suggest that the modulation of SIRT3 by CDK4/6 inhibition might be useful for HCC therapy together with sorafenib, which, unfortunately, has limited efficacy and whose use is often associated with drug resistance.

Nanostructured TiO2 Materials for Analysis of Gout-Related Crystals Using Laser Desorption/Ionization Time-of-Flight (LDI-ToF) Mass Spectrometry.

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 TiO2 materials including TiO2 nanoparticles (P25), TiO2 nanowires synthesized from wet-corrosion method, and the mixture of P25 and TiO2 nanowires (P25/TiO2 nanowires) as inorganic solid matrices. Furthermore, the feasibility of LDI-ToF MS based on these TiO2 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 TiO2 materials has the potential to be a practical approach for the diagnosis of gout and pseudogout.

MALDI-TOF Mass Spectrometry Based on Parylene-Matrix Chip for the Analysis of Lysophosphatidylcholine in Sepsis Patient Sera.

In this work, medical diagnosis of sepsis was conducted via quantitative analysis of lysophosphatidylcholine 16:0 (LPC 16:0) by using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry based on a parylene-matrix chip. In the first step, specific mass peaks for the diagnosis of sepsis were searched by comparing MALDI-TOF mass spectra of sepsis patient sera with healthy controls and pneumonia patient sera. Two mass peaks at m/z = 496.3 and 518.3 were chosen as those that are specifically different for sepsis sera to compare with healthy controls and pneumonia patient sera. These mass peaks were identified to be protonated and sodium adducts of LPC 16:0 by using tandem mass spectra (MS2 and MS3) of purely synthesized LPC 16:0 and extracted LPC 16:0 from a healthy control and a sepsis patient. In the next step, a standard curve for LPC 16:0 for the quantitative analysis of LPC 16:0 with MALDI-TOF MS based on the parylene-matrix chip was prepared, and the statistical correlation to the LC-MS analysis results was demonstrated by using the Bland-Altman test and Passing-Bablok regression. Finally, MALDI-TOF MS based on the parylene-matrix chip was used for the quantification of LPC 16:0 with sera from patients with severe sepsis and septic shock (n = 143), pneumonia patients (n = 12), and healthy sera (n = 31). The sensitivity and the selectivity of medical diagnosis of sepsis was estimated to be 97.9% and 95.5% by using MALDI-TOF MS based on the parylene-matrix chip, respectively.

Gold-nanoparticle-coated magnetic beads for concentration and ionization of analytes for laser desorption/ionization mass spectrometry.

RATIONALE: Magnetic particles coated with gold nanoparticles (Au NPs) (Au-MAGs) were developed and used (1) for sample concentration and (2) as a solid matrix for laser adsorption/desorption mass spectrometry (LDI-MS). METHODS: The Au-MAGs were prepared by (1) coating polystyrene on iron oxide NPs (PS-MNPs), (2) coating poly-l-lysine on the PS-MNPs (PLL-coated PS-MNPs), and (3) coating negatively charged Au NPs on the PLL-coated PS-MNPs (Au-MAGs). RESULTS: The Au-MAGs were used to concentrate the target analyte by means of electrostatic interactions between the positively charged GHP9 and the negatively charged Au-MAGs as well as selective interactions (such as gold-sulfur interactions) between glutathione (GSH) and Au-MAGs. Then, the concentrated analyte could be ionized for LDI-MS. CONCLUSIONS: The Au-MAGs were demonstrated (1) to concentrate the target analyte in a sample solution, tested by electrostatic interactions and selective interactions between gold and sulfur and (2) to ionize the concentrated analyte for LDI-MS.

Parylene-Coated Polytetrafluoroethylene-Membrane-Based Portable Urea Sensor for Real-Time Monitoring of Urea in Peritoneal Dialysate.

A portable urea sensor for use in fast flow conditions was fabricated using porous polytetrafluoroethylene (PTFE) membranes coated with amine-functionalized parylene, parylene-A, by vapor deposition. The urea-hydrolyzing enzyme urease was immobilized on the parylene-A-coated PTFE membranes using glutaraldehyde. The urease-immobilized membranes were assembled in a polydimethylsiloxane (PDMS) fluidic chamber, and a screen-printed carbon three-electrode system was used for electrochemical measurements. The success of urease immobilization was confirmed using scanning electron microscopy, and fourier-transform infrared spectroscopy. The optimum concentration of urease for immobilization on the parylene-A-coated PTFE membranes was determined to be 48 mg/mL, and the optimum number of membranes in the PDMS chamber was found to be eight. Using these optimized conditions, we fabricated the urea biosensor and monitored urea samples under various flow rates ranging from 0.5 to 10 mL/min in the flow condition using chronoamperometry. To test the applicability of the sensor for physiological samples, we used it for monitoring urea concentration in the waste peritoneal dialysate of a patient with chronic renal failure, at a flow rate of 0.5 mL/min. This developed urea biosensor is considered applicable for (portable) applications, such as artificial kidney systems and portable dialysis systems.

Electrochemical Detection of C-Reactive Protein in Human Serum Based on Self-Assembled Monolayer-Modified Interdigitated Wave-Shaped Electrode.

An electrochemical capacitance immunosensor based on an interdigitated wave-shaped micro electrode array (IDWµE) for direct and label-free detection of C-reactive protein (CRP) was reported. A self-assembled monolayer (SAM) of dithiobis (succinimidyl propionate) (DTSP) was used to modify the electrode array for antibody immobilization. The SAM functionalized electrode array was characterized morphologically by atomic force microscopy (AFM) and energy dispersive X-ray spectroscopy (EDX). The nature of gold-sulfur interactions on SAM-treated electrode array was probed by X-ray photoelectron spectroscopy (XPS). The covalent linking of anti-CRP-antibodies onto the SAM modified electrode array was characterized morphologically through AFM, and electrochemically through cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The application of phosphate-buffered saline (PBS) and human serum (HS) samples containing different concentrations of CRP in the electrode array caused changes in the electrode interfacial capacitance upon CRP binding. CRP concentrations in PBS and HS were determined quantitatively by measuring the change in capacitance (ΔC) through EIS. The electrode immobilized with anti-CRP-antibodies showed an increase in ΔC with the addition of CRP concentrations over a range of 0.01-10,000 ng mL-1. The electrode showed detection limits of 0.025 ng mL-1 and 0.23 ng mL-1 (S/N = 3) in PBS and HS, respectively. The biosensor showed a good reproducibility (relative standard deviation (RSD), 1.70%), repeatability (RSD, 1.95%), and adequate selectivity in presence of interferents towards CRP detection. The sensor also exhibited a significant storage stability of 2 weeks at 4 °C in 1× PBS.

A Regenerative Immunoaffinity Layer Based on the Outer Membrane of Z-Domains Autodisplaying E. coli for Immunoassays and Immunosensors.

Through orientation control of antibodies, Z-domains autodisplaying Escherichia coli outer cell membrane (OM) may be utilized to improve the sensitivity and limit of detection (LOD) of immunoassays and immunosensors. A regenerative immunoaffinity layer based on Z-domains autodisplaying E. coli OM was developed for the surface plasmon resonance (SPR) biosensor. Regeneration conditions for the Z-domains autodisplaying E. coli OM-based immunoassays and immunosensors were optimized by varying pH and detergent concentration. An E. coli cell-based HRP immunoassay was tested and validated in three sequential regenerative immunoassays under optimal conditions. The OM of Z-domains autodisplaying E. coli was isolated and coated on the two-dimensional substrate (microplate). The OM-based HRP immunoassay was tested and validated in four regenerative immunoassays. This regenerative OM layer was applied to the SPR biosensor. Z-domains autodisplaying OM layered onto the gold surface of SPR biosensors was developed, and the OM-based regenerative immunoaffinity layer with orientation control was tested using CRP analyte. The SPR biosensor regenerative immunoaffinity layer demonstrated that CRP biosensing was repeated for five regeneration cycles with less than 2% signal difference. Therefore, the newly developed regenerative immunoaffinity layer with antibody orientation control may improve biosensing sensitivity and reduce the cost of medical diagnosis.