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Current tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detect either the constituent nucleic acids/proteins of the viral particles or antibodies specific to the virus, but cannot provide information about viral neutralization by an antibody and the efficacy of an antibody. Such information is important about individuals' vulnerability to severe symptoms or their likelihood of showing no symptoms. We immobilized online SARS-CoV-2 spike (S1) protein and angiotensin-converting enzyme 2 (ACE2) into separate surface plasmon resonance (SPR) channels of a tris-nitrilotriacetic acid (tris-NTA) chip to simultaneously detect the anti-S1 antibody and viral particles in serum samples. In addition, with a high-molecular-weight-cutoff filter, we separated the neutralized viral particles from the free antibody molecules and used a sensing channel immobilized with Protein G to determine antibody-neutralized viral particles. The optimal density of probe molecules in each fluidic channel can be precisely controlled through the closure and opening of the specific ports. By utilizing the high surface density of ACE2, multiple assays can be carried out without regenerations. These three species can be determined with a short analysis time (12 min per assay) and excellent sensor-to-sensor/cycle-to-cycle reproducibility (RSD5%). When coupled with an autosampler, continuous assays can be performed in an unattended manner at a single chip for up to 6 days. Such a sensor capable of assaying serum samples containing the three species at different levels provides additional insights into the disease status and immunity of persons being tested, which should be helpful for containing the SARS-CoV-2 spread during the era of incessant viral mutations.

Ratiometric fluorescence immunoassay is known as a spectrofluorimetric variant for sensitive and quantitative analyses of biomarkers. However, there are few comparative studies between it and the traditional, single-wavelength-based immunoassay. In this work, a ratiometric fluorescence immunoassay was designed via the fluorescence resonance energy transfer (FRET) in a system comprising MnO2 nanosheets, o-phenylenediamine (OPD), and fluorescent carbon nanodots (FCNs). The MnO2 nanosheet oxidizes OPD, producing a fluorescence peak at 580 nm, while FCNs exhibit a fluorescence peak at 490 nm. Under the effect of FRET, the fluorescence at 580 nm increases significantly while the fluorescence at 490 nm is reduced. The FRET effect was confirmed by our fluorescence lifetime measurements. We optimized the experimental conditions that include solution pH, OPD concentration, and reaction time. By electrostatically coating the MnO2 nanosheets with antibody molecules and using the resultant materials as a detection probe, the ratiometric fluorescence sensing was extended to the detection of alpha fetoprotein (AFP) in human serum samples on a 96-well titer plate. Our ratiometric fluorescence immunoassay possesses a good dynamic range (from 0.14 to 130.43 pM). The detection limit was estimated to be 0.13 pM (0.009 ng/mL), which is 7.6-fold lower than that of the single-wavelength-based immunoassay. The measurement results of serum samples from healthy and patient donors are highly comparable to those measured by chemiluminescence for the same donors, demonstrating the viability for serological assays of important cancer markers.

A variety of compounds, including Ni-, Fe-, and Co-containing layered double hydroxides (LDHs), have been explored as catalysts for the oxygen evolution reaction (OER). However, few can meet the in...

Ginnalin A (GA), a polyphenol from the red maple, was reported to be a potential ROS scavenger or an activator of nuclear factor erythroid-2 related factor 2 (Nrf2) in cancer cells. However, whether GA could activate Nrf2 in neuronal cells and the exact mode of action are unknown. We performed molecular docking calculations, which revealed that GA fits well into the five subpockets of the Kelch-like ECH-associated protein1 (Keap1) Kelch domain via hydrogen bonding and hydrophobic interaction. Our cytotoxicity assays demonstrate that pretreating SH-SY5Y cells with 20 μM GA effectively prevents cells from oxidative assault by 6-hydroxydopamine (6-OHDA). Fluorescence imaging indicates that upon the GA pretreatment, Nrf2 dissociates from the Keap1-Nrf2 complex and translocates into nucleus to activate the cellular antixodant system. Real-time qPCR quantification and Western blotting verified that the GA pretreatment elevates NAD(P)H quinone oxidoreductase-1 (NQO1) by more than 4.6-fold, heme oxygenase (HO-1) by about 1.2-fold, and the glutamate-cysteine ligase catalytic (GCLC) subunit by 0.7-fold. The higher antixidant protein levels, along with increased glutathione concentration, decrease intracellular reactive oxygen species and alleviate the 6-OHDA-induced oxidative damage. Silence of Nrf2 abrogates the cytoprotection of the GA pretreatment, confirming that the Keap1/Nrf2-ARE (antioxidant response element) pathway is solely responsible for the GA's biological effects. GA is a promising natural compound for sensitizing neuronal cells' antioxidative defense system to offset oxidative stress, a condition closely linked to the pathogenesis of Parkinson's disease.

We enhanced the sample throughput of microplate-based photothermal detection by using a semicylindrical prism to expand a point laser source to a long beam for illuminating multiple wells. Coupled with four epoxy-coated thermocouples in alignment with wells on a 96-well microplate, four parallel immunoassays of C-reaction protein (CRP) with antibody-conjugated gold nanoparticles can be simultaneously performed. The sample throughput is further increased by mounting the Styrofoam-enclosed microplate onto a translational/elevator stage so that immunoassays and thermocouple rinse/drying cycles can be implemented in a programmed fashion. The automated assay with three rinse/drying cycles takes only 34.5 min for four samples or 8.62 min/sample, whereas the manual mode with a single thermocouple and a point light source requires at least 66 min for just one sample. With careful calibration of the energy distribution of the expanded laser beam and controllable immersion of the thermocouples, excellent well-to-well (RSD = 1.3%) and cycle-to-cycle (RSD = 4.0%) reproducibility can be attained. The temperature changes can be correlated with the CRP concentration by the Langmuir isotherm, and the low limit of detection, 0.52 ng/mL or 4.33 pM, is well below the plasma CRP levels of both healthy people (

Transition metal borides are one of the most promising electrocatalysts for oxygen evolution reaction (OER), but the precise role of boron is still not understood. Herein, we demonstrate experimentally and computationally that boron facilitates OER by modulating the interaction energies of the reaction intermediates. Through a simple chemical reduction in an externally applied magnetic field, one-dimensional (1D) Fe2B nanowires (NWs) can be directly deposited onto a three-dimensional (3D) nickel foam (NF). OER in alkaline solution converts interconnected Fe2B NWs to a thicker and larger network of metaborate- and FeOOH-covered Fe2B NWs. The Fe2B NWs/NF-based catalyst lowers the overpotential to 276 mV at 10 mA cm−2 (normalized to the electrochemical surface area). This intrinsic catalytic activity ranks top among the transition-metal-based compounds. This catalyst is highly stable, showing only 6 mV change in overpotential over continuous testing for 11 h. Such high efficiency is mainly attributed to the catalyst's unique electronic structure, accelerated charge transport, and hydrophilic surface. The remarkable stability stems from the increase in corrosion resistance by the metaborate species in the catalyst.

We demonstrate that arrays of Ni3B/Fe2O3 nanotubes (NTAs) supported by a Fe foil can simultaneously boost the kinetics of the oxygen evolution reaction (OER) and hydrogen evolution reaction (HER) after exposure to visible light radiation (OER and HER overpotentials decreased by 162 and 150 mV at 10 mA cm−2, respectively). With a small band gap, Fe2O3 efficiently harnesses solar energy for the overall photoelectrochemical water splitting. Consequently, a current density of 10 mA cm−2 requires only 1.50 V under one-sun illumination. Surface and spectroscopic analyses indicate that photoholes at the Fe2O3 NTAs transfer to Ni3B, rendering the Ni3B surface easier to oxidize into β-Ni(OH)2, which is ultimately converted into the OER-catalyzing γ-NiOOH species at a relatively low anodic potential. Photoelectrons become localized at the HER-active Ni sites in Ni3B. This work integrates photochemistry with electrocatalysis, affording a new avenue for developing high-performance and cost-effective photoelectrocatalysts for energy conversion applications.

Despite its simplicity and specificity, enzyme-linked immunosorbent assay (ELISA) requires conjugation of the enzyme to an antibody and preservation of enzymatic activity during storage and assay. ...

Polydopamine (PDA)-coated or encapsulating Cu3(PO4)2 (Cu3(PO4)2@PDA) nanosheets were synthesized, allowing the C-reaction protein (CRP) antibody to be attached electrostatically for immunosensing of CRP with simple photothermal detection. The antibody-covered Cu3(PO4)2@PDA nanosheets replace the antibody-conjugated enzyme in the enzyme-linked immunosorbant assays. Owing to the high surface area of the 2-D-structured Cu3(PO4)2@PDA nanosheets and the coabsorption of light in the near-IR spectrum by Cu3(PO4)2 and PDA, a small amount of Cu3(PO4)2@PDA confined in the wells of a titer plate generates an easily detectable temperature change after irradiation at 808 nm. The temperature changes, measured by an inexpensive pen-type thermometer, increased linearly with the analyte concentration from 0.42 to 16 pM. We found that the linear relationship can be fitted by the isotherm derived from responses collected from heterogeneous sensors covered with different ligand or antibody densities. The low detection limit (0.11 pM) is largely due to the attachment of a great number of antibodies onto the flat nanosheets. The antibody-covered Cu3(PO4)2@PDA nanosheets are stable and can be used under conditions that are generally unfavorable to enzymatic activities. The excellent agreement between our results and immunoturbidimetric assays of CRP in serum samples from patients and healthy donors demonstrates its utility for disease diagnosis in clinical settings. This cost-effective, biocompatible, and convenient photothermal immunosensor affords a range of possibilities for detecting diverse protein biomarkers.

Docking on the p53-binding site of murine double minute 2 (MDM2) by small molecules restores p53's tumor-suppressor function. We previously assessed 3244 FDA-approved drugs via “computational conformer selection” for inhibiting MDM2 and p53 interaction. Here, we developed a surface plasmon resonance method to experimentally confirm the inhibitory effects of the known MDM2 inhibitor, nutlin-3a, and two drug candidates predicted by our computational method. This p53/MDM2 interaction displayed a dosage-dependent weakening when MDM2 is pre-mixed with drug candidates. The inhibition efficiency order is nutlin-3a (IC50 = 97 nM) > bepridil (206 nM) > azelastine (307 nM). Furthermore, we verified their anti-proliferation effects on SJSA-1 (wild-type p53 and overexpressed MDM2), SW480 (mutated p53), and SaOs-2 (deleted p53) cancer cell lines. The inhibitory order towards SJSA-1 cell line is nutlin-3a (IC50 = 0.8 μM) > bepridil (23 μM) > azelastine (25 μM). Our experimental results are in line with the computational prediction, and the higher IC50 values from the cell-based assays are due to the requirement of higher drug concentrations to penetrate cell membranes. The anti-proliferation effects of bepridil and azelastine on the cell lines with mutated and deleted p53 implied some p53-independent anti-proliferation effects.

An interference-free photoelectrochemical (PEC) immunoassay was developed for cardiac troponin I (cTnI) detection. Covalent linkage of cTnI antibody to carboxymethylated (CM-) dextran pre-immobilized onto a gold nanoparticles (AuNPs)-modified TiO

Current serological antibody tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require enzyme or fluorescent labels, and the titer well plates cannot be reused. By immobilizing histidine (His)-tagged SARS-CoV-2 spike (S1) protein onto tris‒nitrilotriacetic acid (tris-NTA) sensor and using the early association phase for mass-transfer-controlled concentration determination, we developed a rapid and regenerable surface plasmon resonance (SPR) method for quantifying anti-SARS-CoV-2 antibody. On a five-channel SPR instrument and with optimized S1 protein immobilization density, each of the four analytical channels is sequentially used for multiple measurements, and all four channels can be simultaneously regenerated once they have reached a threshold value. Coupled with a programmable autosampler, each sensor can be regenerated at least 20 times, enabling uninterrupted assays of more than 800 serum samples. The accuracy and speed of our method compare well with those of the enzyme-linked immunosorbent assay (ELISA), and the detection limit (0.057 μg mL

Murine double minute 2 (MDM2) is an oncoprotein mediating the degradation of the tumor suppressor p53 protein. The physiological levels of MDM2 protein are closely related to malignant transformation and tumor growth. In this work, the simultaneous and label-free determination of free and p53-bound MDM2 proteins from sarcoma tissue extracts was conducted using a dual-channel surface plasmon resonance (SPR) instrument. Free MDM2 protein was measured in one fluidic channel covered with the consensus double-stranded (ds)-DNA/p53 conjugate, while MDM2 bound to p53 was captured by the consensus ds-DNA immobilized onto the other channel. To achieve higher sensitivity and to confirm specificity, an MDM2-specific monoclonal antibody (2A10) was used to recognize both the free and p53-bound MDM2 proteins. The resultant method afforded a detection limit of 0.55 pM of MDM2. The amenability of the method to the analysis of free and p53-bound MDM2 proteins was demonstrated for normal and sarcoma tissue extracts from three patients. Our data reveal that both free and total MDM2 (free and bound forms combined) proteins from sarcoma tissue extracts are of much higher concentrations than those from normal tissue extracts and the p53-bound MDM2 protein only constitutes a small fraction of the total MDM2 concentration. In comparison with enzyme-linked immunosorbent assay (ELISA), the proposed method possesses higher sensitivity, is more cost-effective, and is capable of determining free and p53-bound MDM2 proteins in clinical samples.

t‐Darpp (truncated isoform of dopamine‐ and cAMP‐regulated phosphoprotein) is a protein encoded by the PPP1R1B gene and is expressed in breast, colon, esophageal, gastric, and prostate cancers, as well as in normal adult brain striatal cells. Overexpression of t‐Darpp in cultured cells leads to increased protein kinase A activity and increased phosphorylation of AKT (protein kinase B). In HER2+ breast cancer cells, t‐Darpp confers resistance to the chemotherapeutic agent trastuzumab. To shed light on t‐Darpp function, we studied its secondary structure, oligomerization status, metal‐binding properties, and phosphorylation by cyclin‐dependent kinases 1 and 5. t‐Darpp exhibits 12% alpha helix, 29% beta strand, 24% beta turn, and 35% random coil structures. It binds calcium, but not other metals commonly found in biological systems. The T39 site, critical for t‐Darpp activation of the AKT signaling pathway, is a substrate for phosphorylation by cyclin‐dependent kinase 1 and cyclin‐dependent kinase 5. Gel filtration chromatography, sedimentation equilibrium analysis, blue native gel electrophoresis, and glutaraldehyde‐mediated cross‐linking experiments demonstrate that the majority of t‐Darpp exists as a monomer, but forms low levels (

Surface plasmon resonance (SPR) has been well established as a method of choice for label-free kinetic measurements of biomolecular interactions. The conventional approach involves multiple injections of an analyte of different concentrations into a fluidic channel covered with a fixed ligand density. Optimization of the experimental conditions and assessment of the data quality can be complicated by issues such as disruption of the ligand structure by the regeneration step and the limited availability of the sample solution. By sequentially closing fluidic channels on a five-channel SPR instrument, different densities of a ligand can be immobilized and determined in one step. With a subsequent injection of a single sample solution, SPR sensorgrams can be simultaneously collected to yield binding and dissociation rate constants (ka and kd) and dissociation constant (KD) between the ligand and analyte. For biomolecular interactions that obey the Langmuir isotherm, we show that the fidelity of the kinetic d...

Polydopamine (PDA)-coated or encapsulating Cu

The aberrant autoxidation of norepinephrine (NE) in the presence of oxygen, which is accelerated by Fe(III), has been linked to the pathogenesis of the Parkinson's disease (PD). Adenosine triphosphate (ATP), as a neurotransmitter whose release can be stimulated by tissue damage and oxidative stress, is co-stored and often co-released with NE in presynaptic terminals. We have shown previously that ATP inhibits the iron-catalyzed dopamine oxidation, thereby decreasing the production of certain neurotoxins such as 6-hydroxydopamine. Whether ATP plays a similar role in Fe(III)-catalyzed NE oxidation and how it maintains the NE stability have not been investigated. Here, we studied the coordination in a ternary complex among NE, Fe(III), and ATP, and found that Fe(III) is coordinated as a octahedral center by NE and ATP. Voltammetry and mass spectrometry were employed to examine this ternary complex's modulation of the NE autoxidation. NE-Fe(III)-ATP plays a protective role to modulate the autoxidation and Fe(III)-catalyzed oxidation of NE. The ternary complex can be detected in the substantia nigra (SN), locus coeruleus (LC), and striatum regions of C57BL/6 wild-type mice. In contrast, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse brains displayed a significant decrease of the ternary complex in the SN region and an increase in the LC and striatum areas. We posit that the ternary complex is produced by noradrenergic neurons as a protective regulator against neuronal damage and oxidative stress, contributing to the lower vulnerability of LC neurons with respect to that of SN neurons.

A polythiophene-based solar cell (PTSC) is constructed by photoelectrochemically polymerizing thiophene onto an ultrathin compact TiO2 layer (150 nm thick) covered with a sub-monolayer of tethered 3-{5-[ N, N-bis(4-diphenylamino)phenyl]thieno[3,2- b]thiophen-2-yl}-2-cyano-acrylic acid dye (ca. 10% coverage). The influence of morphology and thickness of the PT film on the photocurrent generated by the PTSC was investigated. With a 270 nm thick PT film and 2,2',7,7'-tetrakis( N, N-di(4-methoxyphenyl)amino)-9,9'-spirobifluorene serving as the hole-transport material, the PTSC exhibited a short-circuit current density JSC of 12.90 ± 0.63 mA/cm2, an open-circuit voltage VOC of 0.81 ± 0.01 V, and a fill factor of 0.72 ± 0.01. The high conversion efficiency (7.52 ± 0.58%) of the PTSC is attributed to the controlled PT growth along the ordered and spatially accessible dye molecules at the compact TiO2 layer, which facilitates charge transfer, prevents the hole/electron recombination, and simplifies the polymer solar cell construction with a stable and easily processable material.

Copper is an essential metal in all organisms. Reliably quantifying and identifying the copper content and oxidation state is crucial, since the information is essential to understanding protein structure and function. Chromophoric ligands, such as Bathocuproine (BC) and its water-soluble analog, Bathocuproinedisulfonic acid (BCS), preferentially bind Cu(I) over Cu(II), and therefore have been widely used as optical probes to determine the oxidation state of copper bound by biomolecules. However, the BCS assay is commonly misused, leading to erroneous conclusions regarding the role of copper in biological processes. By measuring the redox potential of Cu(II)-BCS2 and conducting UV-vis absorption measurements in the presence of oxidizable amino acids, the thermodynamic origin of the potential artifacts becomes evident. The BCS assay was improved by introducing a strong Cu(II) chelator EDTA prior to the addition of BCS to prevent interference that might arise from Cu(II) present in the sample. The strong Cu(II) chelator rids of all the potential errors inherent in the conventional BCS assay. Applications of the improved assay to peptides and protein containing oxidizable amino acid residues confirm that free Cu(II) no longer leads to artifacts, thereby resolving issues related to this persistently misused colorimetric assay of Cu(I) in biological systems.

Solar cells constructed with well-ordered polythiophene (PT) or other π-conjugated polymer films are known to yield better photovoltaic characteristics. We recently constructed a PT-based solar cell (PTSC) wherein the photoactive PT film was grown photoelectrochemically along a dye anchor, 3-{5-[N,N-bis(4-diphenylamino)phenyl]thieno[3,2-b]thiophen-2-yl}-2-cyano-acrylic acid (C207) pre-adsorbed onto a compact TiO2 layer. In an effort to understand the effects of other important factors contributing to the PTSC performance, we compared ex-situ and in-situ doping of ordered PT films with three different alkylammonium cations, tetrabutylammonium (TBA+), tetraethylammonium (TEA+), and tetramethylammonium (TMA+). The PT films that have undergone such ex- and in-situ doping both show higher open-circuit voltage (Voc) and short-circuit current density (Jsc) than their pristine counterparts. The optimal conversion efficiency (η) of 7.53 ± 0.58% was achieved via in-situ PT doping with TMA+. The incident photon-to-electron conversion efficiency (IPCE), electrochemical impedance, and electron lifetime measurements all indicate that in-situ doping is less affected by the cation size and can lead to a higher doping level than the ex-situ doping method. The IPCE also benefits from the use of a thin TiO2 layer, as more light reaches the PT film to generate photohole/electron pairs. With the combined use of a thin layer of TiO2 and an ordered PT film containing a large number of dopant ions, the photohole/electron recombination is significantly suppressed.

The Ni2+-histidine (His) chelation yields a more uniform and predicable orientation of immobilized protein molecules than an amine-coupling reaction in surface plasmon resonance (SPR) analyses. However, the gradual dissociation of His-tagged proteins leads to a long and sloped baseline, which adversely affects kinetic studies. Furthermore, as shown in this work for the first time, the strong binding affinity between the histidine-rich Fc domain of immunoglobulin-type antibodies and Ni-nitrilotriacetic acid (NTA) interferes with the kinetic studies of these antibodies and their His-tagged antigens. By performing an amine-coupling reaction immediately after the Ni2+-His chelation, essentially all of the Ni2+-tethered protein molecules can be covalently linked to the carboxyl groups on the underlying carboxymethylated dextran surface. The sequential injections of pH 8.6 phosphate-buffered saline provided additional time to ensure a higher amine coupling efficiency and reverted NHS esters on the protein molecules to carboxyl groups. The application of our method to antibody/antigen interactions is demonstrated with the kinetic analysis of His-tagged t-DARPP protein/anti-t-DARPP interactions. In a separate experiment, the highly efficient immobilization method resulted in a higher immobilization density of His-tagged human carbonic anhydrase (HCA) II, affording accurate kinetic measurements for the binding of 4-carboxybenzenesulfonamide. In addition, the higher HCA II density enhanced the SPR sensitivity, allowing 4-carboxybenzenesulfonamide to be determined with a remarkable detection limit (14 nM).

An enzyme-free titer plate-based colorimetric assay utilizing functionalized mesoporous silica nanoparticles (MSNs) entrapping pH-indicator molecules has been developed. Pores in the silica nanoparticles were functionalized with phenyltrimethyloxysilane so that pH indicator molecules (thymolphthalein or TP in the present case) can be tightly entrapped through π-π conjugation. To detect prostate specific antigen (PSA), the TP-containing MSNs were coated with polyethylenimine (PEI), which favors the attachment of the negatively charged secondary anti-PSA antibody. The entrapped thymolphthalein molecules can be readily released from the pores with a simple addition of alkaline solution. The resultant bifunctional MSNs were used for signal-amplified detection of PSA captured by the primary antibody preimmobilized in the wells of a plate. Our method possesses a wide dynamic range (0.5 to 8000 pg/mL) wherein the adsorption of the bifunctional MSNs obeys a modified Langmuir isotherm. A detection limit (LOD) down to as low as 0.36 pg/mL can be attained. Owing to the size uniformity of the MSNs and the obviation of enzyme molecules employed in the enzyme-linked immunosorbent assay (ELISA), excellent reproducibility (RSD = 1.12%) was achieved. The selective detection of PSA in human serum samples demonstrates the amenability of our method to detect important biomarkers in complex biological samples, whereas the performance of the assay in a titer plate ensures high throughput and obviates the use of expensive instruments. Both of these features are prerequisites for clinical settings wherein a great number of samples need to be analyzed in a timely fashion.

Two six-port injector valves and one selector valve commonly used in flow injection analysis are combined with a surface plasmon resonance (SPR) instrument wherein solutions introduced from the two inlets counter-flow inside the flow cell. The system is versatile as the same or different solutions can be rapidly and repeatedly introduced to the two fluidic channels in series or in parallel. Unlike most commercial SPR instruments employing a single injector valve, solutions separately injected from the two injector valves can be readily exchanged (

Necroptosis has been found to be involved in the pathogenesis of some lung diseases, but its role in hyperoxic acute lung injury (HALI) is still unclear. This study aimed to investigate contribution of necroptosis to the pathogenesis of HALI induced by hyperbaric hyperoxia exposure in a rat model. Rats were divided into control group, HALI group, Nec-1 (necroptosis inhibitor) group and edaravone group. Rats were exposed to pure oxygen at 250 kPa for 6 h to induce HALI. At 30 min before hyperoxia exposure, rats were intraperitoneally injected with Nec-1 or edaravone, and sacrificed at 24 h after hyperoxia exposure. Lung injury was evaluated by histology, lung water to dry ratio (W/D) and bronchoalveolar lavage fluid (BALF) biochemistry; the serum and plasma oxidative stress, expression of RIP1, RIP3 and MLKL, and interaction between RIP1 and RIP3 were determined. Results showed hyperoxia exposure significantly caused damage to lung and increased necroptotic cells and the expression of RIP1, RIP3 and MLKL. Edaravone pre-treatment not only inhibited the oxidative stress in HALI, but also reduced necroptotic cells, decreased the expression of RIP1, RIP3 and MLKL and improved lung pathology. Nec-1 pretreatment inhibited necroptosis and improved lung pathology, but had little influence on oxidative stress. This study suggests hyperoxia exposure induces oxidative stress may activate necroptosis, involving in the pathology of HALI, and strategies targeting necroptosis may become promising treatments for HALI.

Pyrene derivatives are among the most widely used organic fluorescent photoprobes. Many of them are photosensitizers for hole injection. Pyrenes, however, are mostly UV absorbers, limiting their utility for photonic applications. Nitration of pyrene shifts its absorption to the visible region. Conversely, nitration of pyrene that is already derivatized for covalent labeling, produces mixtures of isomers that are challenging to separate. We present a robust procedure for attaining isomerically pure nitropyrenes. NMR analysis provides unequivocal assignments of the regioisomers and of the structures of the disubstituted nitropyrenes. The added substituents negligibly affect the electronic properties of the nitropyrenes. Photoexcited nitropyrenes undergo efficient triplet formation, making them an attractive choice for triplet sensitizers and photooxidants. Hence, facile and reliable preparation of disubstituted nitropyrenes provides venues for exploring their electronic and photonic utility.

t-Darpp is a protein encoded by the PPP1R1B gene and is expressed in breast, colon, esophageal, gastric, and prostate cancers, as well as in normal adult brain striatal cells. Overexpression of t-Darpp in cultured cells leads to increased protein kinase A activity and increased phosphorylation of AKT (protein kinase B). In HER2+ breast cancer cells t-Darpp confers resistance to the chemotherapeutic agent trastuzumab. To shed light on t-Darpp function, we studied its secondary structure, oligomerization status, metal-binding properties, and phosphorylation by cyclin dependent kinases 1 and 5. t-Darpp exhibits 12% alpha helix, 29% beta strand, 24% beta turn and 35% random coil structures. t-Darpp binds to calcium, but not to other metals commonly found in biological systems. The T39 site, critical for t-Darpp activation of the AKT signaling pathway, is a substrate for phosphorylation by cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 5 (CDK5). Gel filtration chromatography, sedimentation equilibrium analysis, blue native gel electrophoresis, and glutaraldehyde-mediated crosslinking experiments demonstrate that the majority of t-Darpp exists as a monomer, but forms low levels (< 3%) of hetero-oligomers with its longer isoform Darpp-32. t-Darpp has a large Stokes radius of 4.4 nm relative to its mass of 19 kDa, indicating that it has an elongated structure.

The ability of peptides to construct specific secondary structures provides a useful function for biomaterial design that cannot be achieved with traditional organic molecules and polymers. Inhibition of amyloid formation is a promising therapeutic approach for the treatment of neurodegenerative diseases. Existing peptide-based inhibitors are mainly derived from original amyloid sequences, which have very limited sequence diversity and activity. It is highly desirable to explore other peptide-based inhibitors that are not directly derived from amyloid sequences. Here, we develop a hybrid high-throughput computational method to efficiently screen and design hexapeptide inhibitors against amyloid-β (Aβ) aggregation and toxicity from the first principle. Computationally screened/designed inhibitors are then validated for their inhibition activity using biophysical experiments. We propose and demonstrate a proof-of-concept of the "like-interacts-like" design principle that the self-assembling peptides are able to interact strongly with conformationally similar motifs of Aβ peptides and to competitively reduce Aβ-Aβ interactions, thus preventing Aβ aggregation and Aβ-induced toxicity. Such a de novo design can also be generally applicable to design new peptide inhibitors against other amyloid diseases, beyond traditional peptide inhibitors with homologous sequences to parent amyloid peptides.

Diverse methods have been developed to reveal biochemical changes in neurological disorders and to detect biomarkers present in clinical samples. Electrochemical biosensors are attractive because of their low costs, high sensitivity, and easiness for miniaturization. They have the potential for both point-of-care and in vivo monitoring. With clever surface modification and effective immobilization of capture ligands, selective detection of neurological biomarkers can be achieved. This review covers select applications of electroanalytical methods/sensors for proteins, peptides, small molecules, metal ions, and neurotransmitters. Designs of various sensors and devices are reviewed and issues to be addressed for clinically viable sensors are discussed.

Lead is a toxic heavy metal whose detoxification in organisms is mainly carried out by its coordination with some metalloproteins such as metallothioneins (MTs). Two Pb–MT complexes, named as Pb7–MT2(I) and Pb7–MT2(II), form under neutral and weakly acidic conditions, respectively. However, the structures of the two complexes, which are crucial for a better understanding of the detoxification mechanism of Pb–MTs, have not been clearly elucidated. In this Work, coordination of Pb2+ with rabbit liver apo–MT2, as well as with the two individual domains (apo−αMT2 and apo−βMT2) at different pH, were studied by combined spectroscopic (UV–visible, circular dichroism, and NMR) and computational methods. The results showed that in Pb7–MT2(I) the Pb2+ coordination is in the trigonal pyramidal Pb–S3 mode, whereas the Pb7–MT2(II) complex contains mixed trigonal pyramidal Pb–S3, distorted trigonal pyramidal Pb–S2O1, and distorted quadrilateral pyramidal Pb–S3O1 modes. The O-donor ligand in Pb7–MT2(II) was identified as the carboxyl groups of the aspartic acid residues at positions 2 and 56. Our studies also revealed that Pb7–MT2(II) has a greater acid tolerance and coordination stability than Pb7–MT2(I), thereby retaining the Pb2+ coordination at acidic pH. The higher flexibility of Pb7–MT2(II) renders it more accessible to lysosomal proteolysis than Pb7–MT2(I). Similar spectral features were observed in the coordination of Pb2+ by human apo-MT2, suggesting a commonality among mammalian MT2s in the Pb2+ coordination chemistry., The pH-dependent structures of two different Pb7−MT2 complexes were revealed. The chemical stabilities and structural flexibility of these two complexes in proteolytic processing were investigated to gain insight into the lead detoxification process involving MTs. The similar structural, chemical, and biological properties between rabbit liver Pb7−MT2(II) and human Pb7−MT2(II) suggest a commonality in the Pb2+ coordination chemistry among mammalian MT2s.

Cleavage of amyloid precursor protein (APP) by the β-site APP cleaving enzyme 1 (BACE1) is a key step in the formation of amyloid beta (Aβ) peptide, the main component of amyloid plaques in Alzheimer's disease (AD). Suppression of BACE1 activity has thus become an efficient way for the treatment of AD. In this study, BACE1 in the absence or presence of BACE1 inhibitors was exposed to the biotinylated peptide substrate-modified electrode. This step was followed by the attachment of ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. Due to the blockage of the BACE1 activity by select inhibitors, well-defined voltammetric peaks of high signal intensity were obtained. However, featureless voltammogram was obtained upon initiating the cleavage reaction. The proposed method is simple, sensitive, and suitable for monitoring of BACE1 activity and screening of BACE1 inhibitors.

Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic cells, which implicates a role of dopamine (DA) in the etiology of PD. A possible DA degradation pathway is the Fe(III)-catalyzed oxidation of DA by oxygen, which produces neuronal toxins as side products. We investigated how ATP, an abundant and ubiquitous molecule in cellular milieu, affects the catalytic oxidation reaction of dopamine. For the first time, a unique, highly stable DA-Fe(III)-ATP ternary complex was formed and characterized in vitro. ATP as a ligand shifts the catecholate-Fe(III) ligand metal charge transfer (LMCT) band to a longer wavelength and the redox potentials of both DA and the Fe(III) center in the ternary complex. Remarkably, the additional ligation by ATP was found to significantly reverse the catalytic effect of the Fe(III) center on the DA oxidation. The reversal is attributed to the full occupation of the Fe(III) coordination sites by ATP and DA, which blocks O2 from accessing the Fe(III) center and its further reaction with DA. The biological relevance of this complex is strongly implicated by the identification of the ternary complex in the substantia nigra of rat brain and its attenuation of cytotoxicity of the Fe(III)-DA complex. Since ATP deficiency accompanies PD and neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) induced PD, deficiency of ATP and the resultant impairment toward the inhibition of the Fe(III)-catalyzed DA oxidation may contribute to the pathogenesis of PD. Our finding provides new insight into the pathways of DA oxidation and its relationship with synaptic activity.

A challenge in coupling ion-pair chromatography (IPC) on-line with electrospray ionization-mass spectrometry (ESI-MS) is that the nonvolatile ion-pair reagent (e.g., alkyl sulfate for amines or tetrabutylammonium for carboxylic acids) in the mobile phase suppresses the ESI-MS signals in the gas phase and their accumulation can clog the MS sampling interface. Consequently, IPC–ESI-MS is conducted either with a volatile ion-pair reagent, which could compromise the analyte separation efficiency, or with a downstream ion-exchange column to rid the ion-pair reagents of the mobile phase. In the latter approach, the limited capacity of ion-exchange columns requires frequent off-line column regeneration, which affects the separation throughput and prohibits long separations from being performed. A dual-valve, dual-ion exchange column interface of IPC–ESI-MS is designed for undisrupted separations and simultaneous column regeneration. Owing to the efficacy in removing the ion-pair reagent, the detection of eluents of monoamine neurotransmitters by an ion trap MS results in the limits of detection of 0.03 μM for dopamine or DA and 0.01 μM for 5-hydroxytryptamine or 5-HT. These values are lower than those obtained with ion trap MS of similar sensitivity when combined with the use of specialized chromatographic columns or sample preconcentration. Excellent reproducibility was attained with repeatedly regenerated ion-exchange columns (RSD = 4–6%) for an extended period of time (RSD < 6% for six days). DA and 5-HT in rat straital extracts were analyzed and our data demonstrate that interferences inherent in the tissues and the ion-pair reagent have been successfully eliminated. This simple interface should be readily amenable to the separation and MS analysis of other types of polar compounds in complex sample media.

Aggregates of amyloid beta (Aβ) peptides are believed to be responsible for the neuropathology of Alzheimer’s disease. In this work, ferrocene (Fc) is attached to the aggregating core of the Aβ peptides, KLVFFAE. Inhibition of Fc-KLVFFAE aggregation by curcumin, a natural compound, was monitored by HPLC-electrochemical detection (HPLC-EC). The Fc oxidation current is dependent on the incubation condition and curcumin can retain Fc-KLVFFAE in its monomeric form. We demonstrate that tagging Fc to KLVFFAE affords a cost-effective and electroactive mimicry of Aβ(142) and HPLC-EC is suitable for sensitive, reproducible, and facile screening of drugs for inhibiting the aggregation of Aβ peptides.

By reducing free and/or weakly complexed Cu 2+ with a Cu 2+ -reducing agent (ascorbic acid in the present study) and detecting the photoluminescence peak of Cu 2 S-covered CdS quantum dots (QDs) at 650 nm, Cu 2+ concentrations ranging from 1 nM to 1 μM can be readily determined. Unlike other related reports, the present method takes advantage of the more efficient chemical reduction of Cu 2+ to Cu + (with respect to the photochemical reduction inherent in CdS QDs) and the facile deposition of Cu 2 S. As a result, a detection limit of 0.5 nM was achieved, which is at least 2–3 orders of magnitude lower than QD-based detection methods. In contrast with other methods requiring sample pretreatment or Cu 2+ -specific ligands capping QDs, the selectivity of the method towards Cu 2+ is excellent. Among a number of metal ions examined, only Cu 2+ causes the red shift of the CdS photoluminescence. A process causing the shift of the CdS photoluminescence was investigated and described. The matrix effect on the photoluminescent behavior of CdS QDs and the amenability of this method for real samples were also studied. Analyses of Cu 2+ in a river water sample and Cu 2+ complexed by amino acids and proteins in cerebrospinal fluids were performed. The latter analysis reveals that our method can differentiate weakly complexed Cu 2+ ions from the more strongly bound ones. This simple method was also demonstrated to be highly sensitive, accurate and reproducible.

Copper coexists with amyloid-β (Aβ) peptides at a high concentration in the senile plaques of Alzheimer’s disease (AD) patients and has been linked to oxidative damage associated with AD pathology. However, the origin of copper and the driving force behind its accumulation are unknown. We designed a sensitive fluorescent probe, Aβ(1–16)(Y10W), by substituting the tyrosine residue at position 10 in the hydrophilic domain of Aβ(1–42) with tryptophan. Upon mixing Cu(II), Aβ(1–16)(Y10W), and aliquots of Aβ(1–42) taken from samples incubated for different lengths of time, we found that the Cu(II) binding strength of aggregated Aβ(1–42) has been elevated by more than two orders of magnitude with respect to that of monomeric Aβ(1–42). Electron paramagnetic spectroscopic measurements revealed that the Aβ(1–42) aggregates, unlike their monomeric form, can seize copper from human serum albumin (HSA), an abundant copper-containing protein in brain and cerebrospinal fluid. The significantly elevated binding strength of the Aβ(1–42) aggregates can be rationalized by a Cu(II) coordination sphere constituted by three histidines from two adjacent Aβ(1–42) molecules. Our work demonstrates that the copper binding affinity by Aβ(1–42) is dependent on its aggregation state and provides new insight into how and why senile plaques accumulate copper in vivo.

Aside from the well-known influence of serotonin (5-hydroxytryptamine, 5-HT) on emotional regulation, more recent investigations have revealed the importance of this monoamine in modulating cognition. Parachlorophenylalanine (PCPA) depletes 5-HT by inhibiting tryptophan hydroxylase, the enzyme required for 5-HT synthesis and, if administered at sufficiently high doses, can result in a depletion of at least 90% of the brain s 5-HT levels. The present study assessed the long-lasting effects of widespread 5-HT depletions on two tasks of cognitive flexibility in Long Evans rats: effort discounting and reversal learning. We assessed performance on these tasks after administration of either 250 or 500 mg/kg PCPA or saline (SAL) on two consecutive days. Consistent with a previous report investigating the role of 5-HT on effort discounting, pretreatment with either dose of PCPA resulted in normal effortful choice: All rats continued to climb tall barriers to obtain large rewards and were not work-averse. Additionally, rats receiving the lower dose of PCPA displayed normal reversal learning. However, despite intact motivation to work for food rewards, rats receiving the largest dose of PCPA were unexpectedly impaired relative to SAL rats on the pretraining stages leading up to reversal learning, ultimately failing to approach and respond to the stimuli associated with reward. High performance liquid chromatography (HPLC) with electrochemical detection confirmed 5-HT, and not dopamine, levels in the ventromedial frontal cortex were correlated with this measure of associative reward learning.

Understanding the structure of mammal Bi-containing metallothionein-2 (Bi-MT2) is of great physiological significance due to the importance of Bi-MT2 in alleviating adverse effect of anti-cancer drugs. A unique feature of rabbit liver Bi-MT2 is the metal-oxygen bond (BiO), which is absent in well-characterized Zn-MT2 and Cd-MT2. However, the ligand contributing to the BiO bonding in Bi-MT2 remains unidentified. In this study, the coordination of Bi(3+) to rabbit liver metal-free metallothionein was investigated using both experimental and theoretical methods. UV-visible and circular dichroism spectra indicate that Bi-MT2 has a different secondary structure from those of Zn-MT2 and Cd-MT2. Three possible Bi(3+) coordination structures in Bi(7)-MT2 and relative binding free energies were calculated using the density functional theory. Absorption spectra corresponding to these coordination structures were evaluated by time-dependent density functional theory. Our computation results are consistent with the UV-vis spectroscopic data and strongly suggest that the carboxyl group in the aspartic acid residues contributes to the BiO bond formation.

MicroRNAs (miRNAs), acting as oncogenes or tumor suppressors in humans, play a key role in regulating gene expression and are believed to be important for developing novel therapeutic treatments and clinical prognoses. Due to their short lengths (17-25 nucleotides) and extremely low concentrations (typicallypicomolar) in biological samples, quantification of miRNAs has been challenging to conventional biochemical methods, such as Northern blotting, microarray, and quantitative polymerase chain reaction (qPCR). In this work, a biotinylated miRNA (biotin-miRNA) whose sequence is the same as that of a miRNA target is introduced into samples of interest and allowed to compete with the miRNA target for the oligonucleotide (ODN) probe preimmobilized onto an electrode. Voltammetric quantification of the miRNA target was accomplished after complexation of the biotin-miRNA with ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. The Fc oxidation current was found to be inversely proportional to the concentration of target miRNA between 10 fM and 2.0 pM. The method is highly reproducible (relative standard deviation (RSD)5%), regenerable (at least 8 regeneration/assay cycles without discernible signal decrease), and selective (with sequence specificity down to a single nucleotide mismatch). The low detection levels (10 fM or 0.1 attomoles of miRNA in a 10 μL solution) allow the direct quantification of miRNA-182, a marker correlated to the progression of glioma in patients, to be performed in serum samples without sample pretreatment and RNA extraction and enrichment. The concentration of miRNA-182 in glioma patients was found to be 3.1 times as high as that in healthy persons, a conclusion in excellent agreement with a separate qPCR measurement of the expression level. The obviations of the requirement of an internal reference in qPCR, simplicity, and cost-effectiveness are other additional advantages of this method for detection of nucleic acids in clinical samples.