Construction of Protein Probe with a His-tag and an Electron-transfer Peptide for a Target Protein Sensing

A protein probe with an electron-transfer peptide and a His-tag was designed to electrochemically sense a target protein. We selected tyrosine-rich (Y₄C) and tryptophan-rich (W₄C) peptides for use as electron-transfer agents. The peak for oxidation was based on the oxidations of the phenolic hydroxy groups in Y₄C and on the indole rings in W₄C. Asialofetuin (ASF) with galactose residues was the protein probe, and a galactose recognition protein, soybean agglutinin (SBA), was the target protein. A protein probe composed of an amino acid and carbohydrate residue was expected to be biocompatible. When voltammetric measurements were performed using a glassy carbon electrode, the oxidation peaks of H₆Y₄C and ASF-H₆Y₄C appeared at the same potential. The peak current of ASF-H₆Y₄C was 4-fold that of H₆Y₄C because of the stronger adsorption of ASF-H₆Y₄C onto the electrode. The electrode response of ASF-H₆Y₄C with SBA was half that of ASF-H₆Y₄C alone. By contrast, the peak current of ASF-Y₄CH₆ was higher than that of ASF-H₆Y₄C, which was the result of a greater degree of contact between the Y₄C moieties and an electrode. On the other hand, the voltammetric behaviors of ASF with W₄C and a His-tag were similar to those with Y₄C and a His-tag. The sensitivity of SBA using ASF-Y₄CH₆ was at the 10⁻¹³ M level. To confirm the function of the sensing system, measurements were performed in human serum with SBA and ASF-Y₄CH₆. When SBA was added, the serum had a concentration that ranged between 5.0×10⁻¹³ and 4.0×10⁻¹² M, and the amount of SBA that could be recovered ranged from 97 to 101%. Consequently, this system could be applied to the detection of SBA in serum.     

Fluorescent Light‐up Probe for the Detection of Protein Aggregates

A fluorescent dye was decorated with water‐soluble pyridinium groups in order to be applied in the detection of cyclodextrins or DNA. The dye displays an enhancement of its emission intensity when the internal rotations are restricted due to the formation of an inclusion complex with cyclodextrins or upon interaction with DNA. In vivo, the fluorescent probe can stain protein aggregates with a selectivity comparable to the widely used Proteostat®.     

Studies on the Toluidine Blue Dimer as a Fluorescence Probe for Protein Assays

Abstract

A spectrofluorometric method was developed for the determination of total serum protein by exploring toluidine blue (TB) as the fluorescence probe. The fluorescence intensity of TB at 648 nm was significantly quenched in the presence of sodium dodecylbenzene sulfonate (SDBS) by forming a dimer of the dye, which can afterwards reconvert to monomer when proteins were added accompanied by the recovery of the fluorescence. This might be attributed to the modulated transferring of the dimer‐monomer equilibrium of TB in the anionic surfactant caused by the addition of protein. A linear calibration graph was obtained in the range of 0.5–50 mg/l BSA, with a detection limit of 0.15 mg/l and a RSD of 1.3% (n=11, 5.0 mg/l BSA). Total proteins in human serums were analyzed by using the present procedure and the results agreed well with those obtained by the Biuret method.     

The Mitochondrial Protein MitoNEET as a Probe for the Allostery of Glutamate Dehydrogenase

The proteins glutamate dehydrogenase (GDH) and mitoNEET are both targets of drug development efforts to treat metabolic disorders, cancer, and neurodegenerative diseases. However, these two proteins differ starkly in the current knowledge about ligand binding sites. MitoNEET is a [2Fe-2S]-containing protein with no obvious binding site for small ligands observed in its crystal structures. In contrast, GDH is known to have a variety of ligands at multiple allosteric sites thereby leading to complex regulation in activity. In fact, while GDH can utilize either NAD(H) or NADP(H) for catalysis at the active site, only NAD(H) binds at a regulatory site to inhibit GDH activity. Previously, we found that mitoNEET forms a covalent bond with GDH in vitro and increases the catalytic activity of the enzyme. In this study we evaluated the effects of mitoNEET binding on the allosteric control of GDH conferred by inhibitors. We examined all effectors using NAD or NADP as the coenzyme to determine allosteric linkage by the NAD-binding regulatory site. We found that GDH activity, in the presence of the inhibitory palmitoyl-CoA and EGCG, can be rescued by mitoNEET, regardless of the coenzyme used. This suggests that mitoNEET rescues GDH by stabilizing the open conformation.     

荧光各向异性法快速测定荧光标记物对蛋白质的标记比

免疫荧光技术是免疫学检测的重要手段之一,该技术在病原微生物的早期诊断、自身免疫研究、抗原或抗体的免疫组化定位等方面都得到了广泛应用^[1].荧光色素对抗体(或抗原)标记比的测定是免疫荧光技术的重要部分.     

Hydrodynamics of bioreactor systems for liquid-liquid contacting

Applied Biochemistry and Biotechnology - Two liquid-liquid bioreactors, a stirred-tank and a novel electrostatic-dispersion system, are being used to investigate biodesulfurization of oil by...     

A New Fluorescein Isothiocyanate-Piperidine Self-ordered Ring Phosphorescent Probe for the Determination of Trace Protein

The ? COOH in fluorescein isothiocyanate (FITC) reacted with ? NH? in piperidine (P) to form FITC‐P on the center of indentation of polyamide membrane (PAM) when drying for 2 min at (92±1)°C. Then, the FITC‐P diffused outward from the indentation center and formed the round SOR‐P‐FITC (containing the FITC‐P self‐ordered rings). Thus, multi‐FITC accumulated on SOR‐P‐FITC, leading to the enhancement of RTP signal on bio‐target, whose I_p increased 2.0 times compared with non‐generated SOR. When bovine serum albumin (BSA) was added to the center of SOR‐P‐FITC, ? NCS of FITC in SOR‐P‐FITC reacted with ? NH₂ of BSA to form SOR‐P‐FITC‐BSA, which caused the RTP signal of FITC to enhance sharply. The ΔI_p of the system was 3.4 times higher than that without β‐CD and 4.0 times higher than that without SOR‐P‐FITC formed. Its ΔI_p was linear to the content of BSA. Therefore, a new solid substrate‐room temperature phosphorimetry (SS‐RTP) for the determination of trace protein was established using SOR‐P‐FITC as a phosphorescent probe. Under the optimum condition, the linear range of this method was 0.040–16.0 ag·spot^?1 with a detection limit (LD) of 8.5 zg·spot^?1 (0.40 µL sample solution per spot, the corresponding concentration was 2.1×10^?17 g·mL^?1), and the regression equation of working curve was ΔI_p=3.848+4.240m_BSA (ag·spot^‐1), n=6, correlation coefficient (r) was 0.9993. This method with high sensitivity had been applied to determining the content of trace protein in the water samples, and the results coincided well with those obtained with pyrocatechol violet‐Mo(VI) method (P.V.M.M.). At the same time, the mechanism of SS‐RTP using SOR‐P‐FITC as a phosphorescent probe (SOR‐P‐FITC‐SS‐RTP) was discussed.     

纳米荧光小球标记在蛋白质微阵列检测中的应用研究

研究了不经分离、一次性制备氨基化联吡啶钌掺杂的双层二氧化硅纳米小球的方法。实验证明该纳米小球尺寸均匀,光稳定性、水溶性好,分散稳定。通过简单的偶联反应后,它能有效的和蛋白质结合,结合后的蛋白能保持其生物活性。以此纳米荧光小球为标记物,应用于蛋白质微阵列的定量检测,结果发现其效果明显优于相同条件下以异硫氰酸荧光素(FITC)为标记物的定量结果,检出限可以达到3.5 mg/L。     

A Supercharged Fluorescent Protein as a Versatile Probe for Homogeneous DNA Detection and Methylation Analysis

Supercharged proteins are a new class of functional proteins with exceptional stability and potent ability to deliver bio‐macromolecules into cells. As a proof‐of‐principle, a novel application of supercharged proteins as a versatile biosensing platform for nucleic acid detection and epigenetics analysis is presented. Taking supercharged green fluorescent protein (ScGFP) as the signal reporter, a simple turn‐on homogenous method for DNA detection has been developed based on the polyionic nanoscale complex of ScGFP/DNA and toehold strand displacement. This assay shows high sensitivity and potent ability to detect single‐base mismatch. Furthermore, combined with bisulfite conversion, this ScGFP‐based assay was further applied to analyze site‐specific DNA methylation status of genomic DNA extracted from real human colon carcinoma tissue sample with ultrahigh sensitivity (4 amol methylated DNA).     

An Azo Coupling Strategy for Protein 3-Nitrotyrosine Derivatization

Herein, a strategy for the selective derivatization of 3-nitrotyrosine-containing proteins using the classic azo coupling reaction as the key step is described. This novel approach featured multiple advantages and was successfully applied to detect picomole levels of protein tyrosine nitration in biological samples.     

An Eosin Y Method for Protein Determination in Solution

ABSTRACT

A method for protein determination using eosin Y is described. Eosin Y reacts with proteins under acidic conditions and forms an intense pink proteineosin Y complex with an absorbance maximum of 535-545nm. The color produced from this reaction is stable and its absorbance increases proportionally over a wide range of protein concentrations (standard assay, 50-1000μg/ml of BSA; microassay, 1.25-50μg/ml of BSA). The eosin Y method can be performed at room temperature without further incubation after vortex mixing. The assay has advantages such as (i) simple procedures, (ii) stable dye-protein color with no precipitation of dye-protein complex for at least 72h, (iii) good reproducibility and (iv) rapid reaction. The characterization of the eosin Y method extends its best application to a routine protein determination in solution.     

An Integrated DNA Modified Dual-microelectrode Sensor Probe

A unique method for preparing a coaxial dual-microelectrode sensor by vaporizing the nano-thickness Au layer on the DNA modified carbon fiber micro-column electrode was illustrated. The dual-electrode showed particular merit for determination in biological systems.     

Hydrodynamics with heated microelectrodes

Voltammetry with continuously heated microelectrodes yields well-defined sigmoidal current-voltage curves. Unlike hydrodynamic electrodes, the surface temperature of a heated electrode can be adjusted arbitrarily within a certain range. Inside a thermal layer, a virtually stagnant diffusion layer of constant thickness _diff exists. _diff is independent of temperature within a certain range. On the basis of this theory, equations are derived which play a similar role to, for instance, the Levich equation for the rotating disk electrode. Reasonable agreement of the derived relationship with the results from voltammetric experiments is obtained.     

双链荧光核酸适体探针用于蛋白质的简便快速检测

发展了一种基于双链荧光核酸适体(F-Aptamer)探针的简单快速检测蛋白质的分析方法.该双链荧光Aptamer探针由一条带荧光标记的Aptamer探针和带猝灭标记的互补DNA组成,当靶蛋白存在时,能形成比双链荧光Aptamer探针更稳定的F-Aptamer/蛋白质复合物,并发出荧光,从而实现对蛋白质的简便快速检测,检测线性范围为6~100 nmol/L,检出限为6 nmol/L.该方法设计简单,对核酸适体分子的大小和空间结构没有要求,可作为一种通用的基于F-Aptamer识别机理的蛋白质检测方法.     

锂离子电池介观尺度光滑粒子水力学模型

针对锂离子电池内耦合电化学反应的多物理传输过程,采用光滑粒子水力学数值技术,开发了可以考虑电极(包括隔膜)介观微结构的数值模型.以电极中固体活性物颗粒尺寸为主要考虑参数,初步探讨了该模型用于电极介观微结构设计的可行性.模型模拟得到放电过程中电池内部Li/Li+浓度场、固/液相电势场以及交换流密度等微观细节分布,以及电池宏观性能如输出电压等,据此可以分析并揭示电池放电过程的基础物理化学机制、电池宏观性能与构成电极的固体活性物颗粒尺寸之间的关联.研究还发现:当阴、阳极固体活性物颗粒尺寸均较小时,固体活性物颗粒内部Li分布更为均匀,电化学反应更均匀发生,电池输出电压最高.     

An Integrated Microfluidic Probe for Mass Spectrometry Imaging of Biological Samples**

Ambient ionization based on liquid extraction is widely used in mass spectrometry imaging (MSI) of molecules in biological samples. The development of nanospray desorption electrospray ionization (nano-DESI) has enabled the robust imaging of tissue sections with high spatial resolution. However, the fabrication of the nano-DESI probe is challenging, which limits its dissemination to the broader scientific community. Herein, we describe the design and performance of an integrated microfluidic probe (iMFP) for nano-DESI MSI. The glass iMFP, fabricated using photolithography, wet etching, and polishing, shows comparable performance to the capillary-based nano-DESI MSI in terms of stability and sensitivity; a spatial resolution of better than 25 μm was obtained in these first proof-of-principle experiments. The iMFP is easy to operate and align in front of a mass spectrometer, which will facilitate broader use of liquid-extraction-based MSI in biological research, drug discovery, and clinical studies.