催化发夹组装用于碱基切除修复酶活性检测研究

碱基切除修复酶在 DNA 损伤修复过程中具有重要作用,且其检测 与癌症等疾病的诊断相关。传统的碱基切除修复酶检测方法操作复杂、灵敏 度低,且仅对酶的浓度进行定量分析而非酶的活性。为此,建立了基于催化 发夹组装介导信号放大用于核酸内切酶 IV（Endo IV）活性的检测方法。该 方法利用 Endo IV 的活性作用于底物探针,将引发序列释放而引发催化发夹 自组装信号放大,以实现 Endo IV 的活性检测分析。通过荧光检测实验可知, 该方法检测下限为 3.7×10 -7 U/mL,可选择性地对 Endo IV 的活性进行检测, 是一种设计简单、操作简便、灵敏度高的碱基切除修复酶活性检测方法。     

Proteolytic Fluorescent Signal Amplification on Gold Nanoparticles for a Highly Sensitive and Rapid Protease Assay

A new strategy for highly sensitive and rapid protease assay is developed by mediating proteolytic formation of oligonucleotide duplexes and using the duplexes for signal amplification. In the presence of matrix metalloprotease‐2 (MMP‐2), fragmentation of the intact DNA–peptide on gold nanoparticles (GNP) by hydrolytic cleavage of a peptide bond within the substrate allows diffusion of DNA away from the GNP and the formation of a DNA/RNA heteroduplex, leading to digestion of RNA by RNase H. Because of the high quenching efficacy of GNP to the fluorophore in RNA and multiple digestions of the RNA, the fluorescence signal recovery is amplified. This method permits the assessment of the activity of MMP‐2 at concentrations as low as 10 pM within 4 h. Compared with the reported protease nanosensors using quantum dots, GNP, and magnetic nanoparticles with the same peptide sequence, the assay time of this method is sixfold faster and the limit of detection is 100‐fold more sensitive. The formulations for proteolytic formations of oligonucleotides duplexes for signal amplification on GNP could lead to the development of more sensitive and rapid protease assay techniques, thus extending the role of proteases as therapeutic targets and disease indicators.     

Carbon Nanotubes Multifunctionalized by Rolling Circle Amplification and Their Application for Highly Sensitive Detection of Cancer Markers

There are still challenges for the development of multifunctional carbon nanotubes (CNTs). Here, a multiwalled carbon nanotube (MWCNT)‐based rolling circle amplification system (CRCAS) is reported which allows in situ rolling circle replication of DNA primer on the surface of MWCNTs to create a long single‐strand DNA (ssDNA) where a large number of nanoparticles or proteins could be loaded, forming a nano‐biohybridized 3D structure with a powerful signal amplification ability. In this strategy, the binding ability of proteins, hybridization, replication ability of DNA, and the catalytical ability of enzymes are integrated on a single carbon nanotube. The CRCAS is then used to develop colorimetric and chemiluminescent assays for the highly sensitive and specific detection of cancer protein markers, alpha‐fetoprotein (AFP) and prostate specific antigen (PSA). The colorimetric CRCAS assay is 4000 times more sensitive than a conventional enzyme‐linked immunosorbent assay (ELISA), and its concentration range is 10 000 times wider. Control experiments show that as low as 10 pg mL^?1 AFP or PSA could be detected even in the presence of interfering protein markers with a more than 10⁵‐fold greater concentration in the sample, demonstrating the high specificity of the CRCAS assay. The limit of detection of the chemiluminescent CRCAS assays for AFP and PSA are 5 fg mL^?1 (70 aM) and 10 fg mL^?1 (0.29 fM), respectively, indicating that the sensitivity is much higher than that of the colorimetric CRCAS assay. Importantly, CRCAS works well with real biological samples.     

Highly Sensitive Detection of Protein Biomarkers with Organic Electrochemical Transistors

The analysis of protein biomarkers is of great importance in the diagnosis of diseases. Although many convenient and low‐cost electrochemical approaches have been extensively investigated, they are not sensitive enough in the detection of protein biomarkers with low concentrations in physiological environments. Here, this study reports a novel organic‐electrochemical‐transistor‐based biosensor that can successfully detect cancer protein biomarkers with ultrahigh sensitivity. The devices are operated by detecting electrochemical activity on gate electrodes, which is dependent on the concentrations of proteins labeled with catalytic nanoprobes. The protein sensors can specifically detect a cancer biomarker, human epidermal growth factor receptor 2, down to the concentration of 10^?14 g mL^?1, which is several orders of magnitude lower than the detection limits of previously reported electrochemical approaches. Moreover, the devices can successfully differentiate breast cancer cells from normal cells at various concentrations. The ultrahigh sensitivity of the protein sensors is attributed to the inherent amplification function of the organic electrochemical transistors. This work paves a way for developing highly sensitive and low‐cost biosensors for the detection of various protein biomarkers in clinical analysis in the future.     

超灵敏检漏的实时校准

超灵敏检漏的实时校准是国际上尚未解决且未标准化的一个难题,本文利用自建的微流量参考漏率系统对商用检漏仪和超灵敏检漏系统进行了实时校准的实验研究,在10-14Pa.m3/s~10-10Pa.m3/s宽范围漏率内实现了对检漏仪器的实时校准。     

A Sensitive Aptasensor Based on a Hemin/G‐Quadruplex‐Assisted Signal Amplification Strategy for Electrochemical Detection of Gastric Cancer Exosomes

Emerging evidence indicates that exosomes derived from gastric cancer cells enhance tumor migration and invasion through the modulation of the tumor microenvironment. However, it remains a major problem to detect cancer‐specific exosomes due to technical and biological challenges. Most of the methods reported could not achieve efficient detection of tumor‐derived exosomes in the background of normal exosomes. Herein, a label‐free electrochemical aptasensor is presented for specific detection of gastric cancer exosomes. This platform contains an anti‐CD63 antibody modified gold electrode and a gastric cancer exosome specific aptamer. The aptamer is linked to a primer sequence that is complementary to a G‐quadruplex circular template. The presence of target exosomes could trigger rolling circle amplification and produce multiple G‐quadruplex units. This horseradish peroxidase mimicking DNAzyme could catalyze the reduction of H₂O₂ and generate electrochemical signals. This aptasensor exhibits high selectivity and sensitivity toward gastric cancer exosomes with a detection limit of 9.54 × 10² mL^?1 and a linear response range from 4.8 × 10³ to 4.8 × 10⁶ exosomes per milliliter. Therefore, this electrochemical aptasensor is expected to become a useful tool for the early diagnosis of gastric cancer.     

Rapid, Sensitive Detection of Neurotransmitters at Microelectrodes Modified with Self-assembled SWCNT Forests

Carbon nanotube (CNT) modification of microelectrodes can result in increased sensitivity without compromising time response. However, dip coating CNTs is not very reproducible and the CNTs tend to lay flat on the electrode surface which limits access to the electroactive sites on the ends. In this study, aligned CNT forests were formed using a chemical self-assembly method, which resulted in more exposed CNT ends to the analyte. Shortened, carboxylic acid functionalized single-walled CNTs were assembled from a dimethylformamide (DMF) suspension onto a carbon-fiber disk microelectrode modified with a thin iron hydroxide-decorated Nafion film. The modified electrodes were highly sensitive, with 36-fold higher oxidation currents for dopamine using fast-scan cyclic voltammetry than bare electrodes and 34-fold more current than electrodes dipped in CNTs. The limit of detection (LOD) for dopamine was 17 ± 3 nM at a 10 Hz repetition rate and 65 ± 7 nM at 90 Hz. The LOD at 90 Hz was the same as a bare electrode at 10 Hz, allowing a 9-fold increase in temporal resolution without a decrease in sensitivity. Similar increases were observed for other cationic catecholamine neurotransmitters, and the increases in current were greater than for anionic interferents such as ascorbic acid and 3,4-dihydroxyphenylacetic acid (DOPAC). The CNT forest electrodes had high sensitivity at 90 Hz repetition rate when stimulated dopamine release was measured in Drosophila . The sensitivity, temporal resolution, and spatial resolution of these CNT forest modified disk electrodes facilitate enhanced electrochemical measurements of neurotransmitter release in vivo.     

Development of xMAP Assay for Detection of Six Protein Toxins

xMAP technology was used for simultaneous identification of six protein toxins (staphylococcal enterotoxins A and B, cholera toxin, ricin, botulinum toxin A, and heat labile toxin of E. coli). Monoclonal antibody-conjugated xMAP microspheres and biotinilated monoclonal antibodies were used to detect the toxins in a sandwich immunoassay format. The detection limits were found to be 0.01 ng/mL for staphylococcal enterotoxin A, cholera toxin, botulinum toxin A, and ricin in model buffer (PBS-BSA) and 0.1 ng/mL for staphylococcal enterotoxin B and LT. In a complex matrix, such as cow milk, the limits of detection for staphylococcal enterotoxins A and B, cholera toxin, botulinum toxin A, and ricin increased 2- to 5-fold, while for LT the detection limit increased 30-fold in comparison with the same analysis in PBS-BSA. In the both PBS-BSA and milk samples, the xMAP test system was 3-200 times (depending on the toxin) more sensitive than ELISA systems with the same pairs of monoclonal antibodies used. The time required for a simultaneous analysis of six toxins using the xMAP system did not exceed the time required for ELISA to analyze one toxin. In the future, the assay may be used in clinical diagnostics and for food and environmental monitoring.     

Sensitive Detection of Human Insulin Using a Designed Combined Pore Approach

A unique combined pore approach to the sensitive detection of human insulin is developed. Through a systematic study to understand the impact of pore size and surface chemistry of nanoporous materials on their enrichment and purification performance, the advantages of selected porous materials are integrated to enhance detection sensitivity in a unified two‐step process. In the first purification step, a rationally designed large pore material (ca. 100 nm in diameter) is chosen to repel the interferences from nontarget molecules. In the second enrichment step, a hydrophobically modified mesoporous material with a pore size of 5 nm is selected to enrich insulin molecules. A low detection limit of 0.05 ng mL^?1 in artificial urine is achieved by this advanced approach, similar to most antibody‐based analysis protocols. This designer approach is efficient and low cost, and thus has great potential in the sensitive detection of biomolecules in complex biological systems.     

Electrical Graphene Aptasensor for Ultra‐Sensitive Detection of Anthrax Toxin with Amplified Signal Transduction

Detection of the anthrax toxin, the protective antigen (PA), at the attomolar (aM) level is demonstrated by an electrical aptamer sensor based on a chemically derived graphene field‐effect transistor (FET) platform. Higher affinity of the aptamer probes to PA in the aptamer‐immobilized FET enables significant improvements in the limit of detection (LOD), dynamic range, and sensitivity compared to the antibody‐immobilized FET. Transduction signal enhancement in the aptamer FET due to an increase in captured PA molecules results in a larger 30 mV/decade shift in the charge neutrality point (V_g,min) as a sensitivity parameter, with the dynamic range of the PA concentration between 12 aM (LOD) and 120 fM. An additional signal enhancement is obtained by the secondary aptamer‐conjugated gold nanoparticles (AuNPs‐aptamer), which have a sandwich structure of aptamer/PA/aptamer‐AuNPs, induce an increase in charge‐doping in the graphene channel, resulting in a reduction of the LOD to 1.2 aM with a three‐fold increase in the V_g,min shift.     

基于金纳米粒子比色检测重金属铅离子

目的研究一种新的基于半胱氨酸修饰的金纳米粒子的检测重金属铅离子的方法。方法采用柠檬酸钠还原法合成粒径为13 nm的金纳米粒子,并用半胱氨酸进行修饰(Cys-Au NPs)。在Pb2+诱导下,Cys能通过螯合配体结合作用捕获Pb2+,导致Au NPs聚集,使Au NPs特征峰的位置和强度发生变化,且溶液颜色发生变化。结果该比色法的检测范围为0.02~5μmol/L,检出限为0.01μmol/L。通过对比Pb2+与其他金属离子的紫外可见光谱,证明该方法特异性好。结论该比色法可用于Pb2+的实际检测应用中。     

基于敏感区域多结构元素形态学边缘检测算法

在边缘检测算法的应用中,很难同时兼顾图像处理效果和处理速度,为此,提出了一种新的边缘检测算法,将边缘处理集中在感兴趣的图像区域中.该算法利用梯度算子获得图像中的敏感区域,再构造多种结构元素,结合形态学梯度和OTSU分割法检测敏感区域的边缘.应用于沥青路面裂缝图像检测,实验结果表明,与其它边缘检测算法相比,该算法不仅具有很好的边缘提取能力,而且具有很强的抗噪能力.在保证处理效果的同时,也保证了处理速度,有很高的实用性和推广性.     

Amplification of Resonant Rayleigh Light Scattering Response Using Immunogold Colloids for Detection of Lysozyme

A strategy for attomolar‐level detection of small molecule‐size proteins is reported based on Rayleigh light scattering spectroscopy of individual nanoplasmonic aptasensors by exploiting the outstanding characteristics of gold colloids to amplify the nontransparent resonant signal at ultralow analyte concentrations. The fabrication method utilizes thiol‐mediated adsorption of a DNA aptamer on the immobilized Au nanoparticle surface, the interfacial binding characteristics of the aptamer with its target molecules, and the antibody–antigen interaction through plasmonic resonance coupling of the Au nanoparticles. Using lysozyme as a model analyte for disease detection, the detection limit of the aptasensor is ～7 × 10³ aM, corresponding to the LSPR λ_max shift of ～2.25 nm. Up to a 380% increase in the localized resonant λ_max shift is demonstrated upon antibody binding to the analyte compared to the primary response during signal amplification using immunogold colloids. This enhancement leads to a limit of detection of ～7 aM, which is an improvement of three orders of magnitude. The results demonstrate substantial promise for developing coupled plasmonic nanostructures for ultrasensitive detection of various biological and chemical analytes.     

一种基于磁富集靶序列PCR的扩增方法 及其灵敏度检测

以亲和素修饰的磁性纳米颗粒γ-Fe2O3为载体,提出了基于磁富集靶序列PCR扩增方法。首先将结合有生物素标记特异性引物的靶序列富集到亲和素修饰的γ-Fe2O3纳米颗粒表面,然后通过变性获取单链的靶序列,再进行PCR扩增。同时,优化了靶序列和特异性引物杂交的最适温度和磁性纳米颗粒γ-Fe2O3的最佳用量,并对该方法的灵敏度进行了检测。通过实验得出:该方法中,最适的杂交温度为53℃,磁性纳米颗粒的最佳用量为90μg,靶序列的最低检出浓度为5×10-10ng/mL。     

An Intensity-Stabilized Diode-Laser Spectrometer for Sensitive Detection of NH3

This paper reports on a compact analyzer for ammonia monitoring in the air based on near-infrared wavelength-modulation diode-laser absorption spectroscopy. Particular attention is devoted to the problem of residual amplitude modulation (RAM), which is responsible for the occurrence of the line profiles' distortions. The RAM effects are eliminated by means of an intensity-control feedback loop based on an acoustooptic modulator. This offers the advantage of simplifying enormously the spectral-analysis procedure, which, otherwise, would require a considerable amount of calculation if the RAM distortions were considered     

New Nanoplatform based on Upconversion Nanoparticles for Sensitive Detection of Acute promyelocytic Leukemia

正Acute promyelocytic leukemia(APL)is a special leukemia accounting for above 10%of adult acute myeloid leukemia.The t(15;17)(q22;q21)is a specific chromosome reciprocal translocation of APL,resulting in the generation of a fusion gene between a retinoic acid receptor alpha(RARα)and promyelocytic leukemia(PML)at the molecular level.Many research demonstrate that PML/RARa fusion gene can be regarded as the