Novel electrochemical biosensor based on functional composite nanofibers for sensitive detection of p53 tumor suppressor gene

A novel electrochemical biosensor based on functional composite nanofibers for sensitive hybridization detection of p53 tumor suppressor using methylene blue (MB) as an electrochemical indicator is developed. The carboxylated multi-walled carbon nanotubes (MWNTs) doped nylon 6 (PA6) composite nanofibers (MWNTs–PA6) was prepared using electrospinning, which served as the nanosized backbone for pyrrole (Py) electropolymerization. The functional composite nanofibers (MWNTs–PA6–PPy) used as supporting scaffolds for ssDNA immobilization can dramatically increase the amount of DNA attachment and the hybridization sensitivity. The biosensor displayed good sensitivity and specificity. The target wild type p53 sequence (wtp53) can be detected as low as 50 fM and the discrimination is up to 57.5% between the wtp53 and the mutant type p53 sequence (mtp53). It holds promise for the early diagnosis of cancer development and monitoring of patient therapy.     

Carbon nanotube field effect transistor biosensor for the detection of toxins in seawater

Disposable field effect transistors (FET) biosensors (bio-FET) based on carbon nanotubes were fabricated for detection of domoic acid (DA), which belongs to the group of biotoxins associated with the amnesic shellfish poisoning. The analytical results obtained with the bio-FET were compared with those obtained with a traditional methodology (enzyme-linked immunosorbent assay) in order to validate the bio-FET for DA detection. Standard solutions of DA with concentrations between 10 and 500 ng L^?1 were tested in order to construct the calibration curve, where five bio-FET were used for reproducibility estimation and two analytical measurements were performed for each bio-FET for repeatability estimation. Ten spiked artificial seawater samples were used to validate the bio-FET. The obtained reproducibility (0.52–1.43%), repeatability (0.57–1.27%), limit of detection (10 ng L^?1) and recovery range (92.3–100.3%) reveal an adequate analytical performance of the bio-FET for the detection of DA in environmental samples such as seawater samples.     

A novel method for the sensitive detection of mutant proteins using a covalent-bonding tube-based proximity ligation assay

Tumorigenesis is the cumulative result of multiple gene mutations. The mutant proteins that are expressed by mutant genes in cancer cells are secreted into the blood and are useful biomarkers for the early diagnosis of cancer. However, some difficulties exist; for example, the same gene will express different protein mutants in different patients, and early tumors secrete only small amounts of mutant protein. Thus, the presence of mutant proteins in plasma has not previously been exploited for the early diagnosis of cancer. Proximity ligation assay is a protein-detection method that has been developed in recent years and has been widely used because of its high sensitivity. However, this approach still suffers from some shortcomings that should be addressed. In this paper, we develop a covalent-bonding tube-based proximity ligation assay (TB-PLA). The limit of detection of TB-PLA for 0.001 pM, and the method exhibited a broad dynamic range of up to seven orders of magnitude. Furthermore, we coupled the conformation-specific antibody PAb240 of p53 mutants to PCR tubes for TB-PLA. The assay was capable of detecting an approximately 500-fold lower concentration of mutant p53 in serum compared with sandwich ELISA. Thus, we demonstrate TB-PLA to be a highly sensitive and effective approach that is suitable for the early clinical diagnosis of cancer using the conformation-specific antibodies of protein mutants.     

Multiplexed p53 Mutation Detection by Microchip Electrophoresis with Laser-Induced Fluorescence Detector

A form of single‐strand DNA‐conformation polymorphism analysis (SSCP) employing nondenaturing slab gel electrophoresis is applicable to the genetic diagnosis of mutations at exons 7, 8 and 9 of the p53 gene. Recently, microchip electrophoresis (ME) systems have been used in SSCP analysis instead of conventional slab gel electrophoresis in terms of speed, sensitivity and automation. The aim of the present study was to investigate the application of SSCP and ME analysis as a rapid and effective method to the detection of mutations for exons 7, 8 and 9 of the p53 gene. It was found that using the electric field strength 260 V/cm and the sieving matrix of 4 mg/mL poly(ethylene oxide) was very useful to achieve better resolution and fast detection of mutations at exons 7, 8 and 9 of p53 gene. Under the optimized conditions, mutations at exons 7–9 of p53 gene were analyzed within 60 s and the relative standard deviation values of the migration times were less than 5.81% (n=5). The detection limit can be as low as 1 ng·L^?1.     

Synthesis and Biological Evaluation of Novel Synthetic Indolone Derivatives as Anti-Tumor Agents Targeting p53-MDM2 and p53-MDMX

A series of novel indolone derivatives were synthesized and evaluated for their binding affinities toward MDM2 and MDMX. Some compounds showed potent MDM2 and moderate MDMX activities. Among them, compound A13 exhibited the most potent affinity toward MDM2 and MDMX, with a K_i of 0.031 and 7.24 μM, respectively. A13 was also the most potent agent against HCT116, MCF7, and A549, with IC₅₀ values of 6.17, 11.21, and 12.49 μM, respectively. Western blot analysis confirmed that A13 upregulated the expression of MDM2, MDMX, and p53 by Western blot analysis. These results indicate that A13 is a potent dual p53-MDM2 and p53-MDMX inhibitor and deserves further investigation.     

基于金胶的选择性聚集实现p53基因的电化学超灵敏检测

介绍了一种利用金胶的选择性聚集实现信号扩增的超灵敏的电化学方法, 用于人类p53肿瘤抑制剂基因的检测. 在实验中, 根据p53基因的序列设计了能特异性检测p53肿瘤抑制剂基因的二段探针, 在一段探针上固定磁性颗粒以捕获并富集目标基因, 同时在另一段探针上标记金纳米颗粒作为检测信标. 另外, 通过硫代三聚氰酸和金纳米颗粒的自组装作用, 形成金纳米颗粒和硫代三聚氰酸的网状结构, 获得金纳米颗粒的选择性聚集, 实现信号扩增. 用此法检测目标p53野生型DNA, 最低检测限为2.24×10-17 mol/L, 同时进一步研究了该探针对p53野生型和一碱基错配的突变型的选择性.     

Characterization of facilitated diffusion of tumor suppressor p53 along DNA using single-molecule fluorescence imaging

Sequence-specific DNA-binding proteins can maintain and regulate cellular functions by accurately and quickly binding to target sequences among large amounts of nontarget DNA. The facilitated diffusion mechanism of DNA-binding proteins—a combination of three-dimensional (3D) diffusion and one-dimensional (1D) sliding along DNA—has been proposed to explain the target binding accuracy and rapidity and has been partially confirmed experimentally. Nonetheless, quantitative elucidation of the mechanism has remained difficult. Furthermore, many additional steps in facilitated diffusion have been proposed. In this review, we introduce the theoretical and experimental studies and the current understanding of facilitated diffusion of DNA-binding proteins. We focused on tumor suppressor p53 as a key protein subject to facilitated diffusion; p53 regulates various cellular processes such as cell cycle arrest, DNA repair, and apoptosis upon binding to a target sequence of DNA after activation by external stress to the cell. We describe the research on the 3D diffusion and 1D sliding of p53 mainly via single-molecule fluorescence microscopy. In addition to the demonstration of the 1D sliding of p53, recent experiments revealed multiple modes of 1D sliding, regulation of the target recognition, and the constant search distance despite changes in the concentrations of divalent cations. Furthermore, rotation-coupled 1D sliding along DNA is suggested. A comparison of parameters of the facilitated diffusion of p53 and those of other DNA-binding proteins characterized so far suggests that the ratio of 3D diffusion and 1D sliding is close to the theoretical optimum of 1:1 for several proteins including p53.     

Detection of microcystins in environmental samples using surface plasmon resonance biosensor

An indirect inhibitive surface plasmon resonance (SPR) immunoassay was developed for the microcystins (MCs) detection. The bioconjugate of MC-LR and bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. A serial premixture of MC-LR standards (or samples) and monoclonal antibody (mAb) were injected over the functional sensor surface, and the subsequent specific immunoreaction was monitored on the BIAcore 3000 biosensor and generated a signal with an increasing intensity in response to the decreasing MCs concentration. The developed SPR immunoassay has a wide quantitative range in 1-100 μg L⁻¹. Although not as sensitive as conventional enzyme-linked immunosorbent assay (ELISA), the SPR biosensor offered unique advantages: (1) the sensor chip could be reusable without any significant loss in its binding activity after 50 assay-regeneration cycles, (2) one single assay could be accomplished in 50 min (including 30-min preincubation and 20-min BIAcore analysis), and (3) this method did not require multiple steps. The SPR biosensor was also used to detect MCs in environmental samples, and the results compared well with those obtained by ELISA. We conclude that the SPR biosensor offers outstanding advantages for the MCs detection and may be further developed as a field-portable sensor for real-time monitoring of MCs on site in the near future.     

新抗癌基因p16的分析检测研究进展

p16基因又称多肿瘤抑癌基因,其直接参与细胞周期调控,并负向调节细胞的增殖及分裂。研究显示,50%的人类肿瘤细胞株中存在纯合子缺失和突变,认为p16是比p53更为重要的一种新型抗癌基因。p16作为细胞周期中的"刹车装置",一旦失灵会导致细胞的恶性增殖,从而引起恶性肿瘤的发生。本文主要对近年来p16基因相关分析检测技术的原理、方法及进展作一简要综述。     

Electrochemical DNA Hybridization Assay: Enzyme‐Labeled Detection of Mutation in p53 Gene

This paper discusses a new electrochemical DNA hybridization sensing approach based on the detection of a linked enzyme label. In this method we employ enzyme that is attached to a tethered ssDNA oligomer on the surface and the target analyte is a complementary ssDNA oligomer that does not require any pre‐treatment. The advantage of using of enzyme label is in its amplification of the registration of the hybridization event due to the catalytic reaction facilitated in the process. One particular novelty is associated with the use of enzymes that directly communicate with the electrode surface thus allowing for minimizing the need of additional reagents in the assay. The electrochemical assay was demonstrated when using mixed self‐assembled monolayers from thiolated oligonucleotide and 6‐mercapto 1‐hexanol on gold surfaces. Horseradish peroxidase (HRP) is attached to the surface tethered oligonucleotide using streptavidin‐biotin chemistry, and the enzyme successfully established direct electron transfer (DET) with the electrode or mediated electron transfer (MET) using a mediator. Hybridization results in increasing the angle of contact between electrode and DNA and also the stiffness of the ds DNA, which results in displacing the enzyme away from the electrode surface, and thereby reducing the occurrence of direct electron transfer between the enzyme and the electrode. The cyclic voltammetry showed a clear distinction in response between the complete complementary sequence and the two‐base mismatch sequence. Ellipsometric measurements show that the thickness of the thiol modified oligonucleotide on gold surfaces changes before and after hybridization for the complementary sequence, where as a minimal change in thickness was observed for the noncomplementary sequence. The model target analyte in this study was TP53 gene where a specific mutation is a marker for a list of cancers. Mutations of the TP53 gene have been demonstrated in tumors of the colon, breast, lung, ovary, bladder, and many other organs. Analysis of p53 mutations may provide useful information for the diagnosis, prognosis and therapy of cancer.     

The Mechanism of p53 Rescue by SUSP4

p53 is an important tumor‐suppressor protein deactivation of which by mdm2 results in cancers. A SUMO‐specific protease 4 (SUSP4) was shown to rescue p53 from mdm2‐mediated deactivation, but the mechanism is unknown. The discovery by NMR spectroscopy of a “p53 rescue motif” in SUSP4 that disrupts p53‐mdm2 binding is presented. This 29‐residue motif is pre‐populated with two transient helices connected by a hydrophobic linker. The helix at the C‐terminus binds to the well‐known p53‐binding pocket in mdm2 whereas the N‐terminal helix serves as an affinity enhancer. The hydrophobic linker binds to a previously unidentified hydrophobic crevice in mdm2. Overall, SUSP4 appears to use two synergizing modules, the p53 rescue motif described here and a globular‐structured SUMO‐binding catalytic domain, to stabilize p53. A p53 rescue motif peptide exhibits an anti‐tumor activity in cancer cell lines expressing wild‐type p53. A pre‐structures motif in the intrinsically disordered proteins is thus important for target recognition.     

Detection of p53 Gene by Using Genomagnetic Assay Combined with Carbon Nanotube Modified Disposable Sensor Technology

Electrochemical monitoring of DNA hybridization related to p53 gene sequence was investigated using genomagnetic assay combined with single walled carbon nanotube (SWCNT) modified pencil graphite electrodes (PGEs). The hybridization was performed either at magnetic beads (MB) surface or in solution. The enhanced guanine signal was obtained using SWCNT‐PGEs compared to one obtained by unmodified PGEs. The selectivity of genomagnetic assay was tested under optimum conditions. The DLs were calculated as 0.88 µM and 0.11 µM for hybridization performed at MB surface and solution, respectively. This selective, practical and cost effective genomagnetic assay combined with SWCNT‐PGEs is reported herein for the first time.     

Graphene-based immunosensor for electrochemical quantification of phosphorylated p53 (S15)

We reported a graphene-based immunosensor for electrochemical quantification of phosphorylated p53 on serine 15 (phospho-p53¹⁵), a potential biomarker of gamma-radiation exposure. The principle is based on sandwich immunoassay and the resulting immunocomplex is formed among phospho-p53 capture antibody, phospho-p53¹⁵ antigen, biotinylated phospho-p53¹⁵ detection antibody and horseradish peroxidase (HRP)-labeled streptavidin. The introduced HRP results in an electrocatalytic response to reduction of hydrogen peroxide in the presence of thionine. Graphene served as sensor platform not only promotes electron transfer, but also increases the surface area to introduce a large amount of capture antibody, thus increasing the detection sensitivity. The experimental conditions including blocking agent, immunoreaction time and substrate concentration have been optimized. Under the optimum conditions, the increase of response current is proportional to the phospho-p53¹⁵ concentration in the range of 0.2–10 ng mL⁻¹, with the detection limit of 0.1 ng mL⁻¹. The developed immunosensor exhibits acceptable stability and reproducibility and the assay results for phospho-p53¹⁵ are in good correlation with the known values. This easily fabricated immunosensor provides a new promising tool for analysis of phospho-p53¹⁵ and other phosphorylated proteins.     

Indigo Carmine as New Label in PNA Biosensor for Detection of Short Sequence of p53 Tumor Suppressor Gene

A new electrochemical PNA hybridization biosensor for detection of a 15‐mer sequence unique to p53 using indigo carmine (IC) as an electrochemical detector is described in this work. This genosensor is based on the hybridization of target oligonucleotide with its complementary probe immobilized on the gold electrode by self‐assembled monolayer formation. Because this label is electroactive in acidic medium, the interaction between IC and short sequence of p53 is studied by differential pulse voltammety (DPV) in 0.1 M H₂SO₄. The results of electrochemical impedance spectroscopy and cyclic voltammetry in the solution of [Fe(CN)₆]^3?/4? shows no breakage in PNA‐DNA duplex. A decrease in the voltammetric peak currents of IC is observed upon hybridization of the probe with the target DNA. The influence of probe concentration on effective discrimination against non‐complementary oligonucleotides is investigated and a concentration of 10^?7 M is selected. The diagnostic performance of the PNA sensor is described and the detection limit is found to be 4.31×10^?12 M.     

p53-MDM2结合抑制剂药效团模型的构建

采用Catalyst软件, 选择5类共24个p53-MDM2结合抑制剂作为训练集, 经计算机建模、构象优化, 由Catalyst系统构建出药效团模型, 并对药效团进行有效性分析, 结合已知的p53-MDM2结合抑制剂的结构信息, 筛选得到含有一个芳环中心、三个疏水中心和一个氢键受体的具有较好预测能力(Correl=0.941, Config=17.530, 吟cost=150.830)的药效团模型.     

Design and synthesis of functionalized trisaccharides as p53-peptide mimics

Oligosaccharides represent potentially useful scaffolds for the development of peptidomimetics. We report here the design and synthesis of functionalized trisaccharides modeled after an α-helical 15-mer peptide region of p53 which binds to its cellular regulator MDM2. The trisaccharide scaffold was obtained efficiently by applying the sulfoxide glycosylation reaction as a key methodology.