免标记DNA电化学传感器测定凝血酶

研制了一种基于介孔二氧化硅负载纳米金和适体DNA的免标记电化学传感器,用于检测凝血酶的含量。实验中先用三甲基氯硅烷(TMCS)封闭介孔二氧化硅(MPS)前驱体外壁硅羟基的活性,煅烧除去模板剂后,再用氨基丙基三乙氧基硅烷(APTS)与孔道内壁硅羟基反应,接枝氨基,最后得到内壁修饰氨基的介孔硅材料(APTS-TMCS-MPS)。金纳米粒子(GNPs)通过与APTS-TMCS-MPS氨基的静电作用力组装到介孔孔道内,然后和巯基修饰的适体DNA通过金硫键相结合,制得免标记探针(DNA/GNPs/APTSTMCS-MPS)。将探针修饰在玻碳电极表面,制得免标记DNA电化学传感器。将该传感器与含有凝血酶的待测液温育反应后,凝血酶和固定在介孔内的适体DNA发生特异性反应,形成位阻较大的适体DNA和凝血酶的复合物,从而增加了介孔孔道内的空间位阻,导致电流响应信号降低。随着凝血酶浓度的增加,孔道内部的位阻也随之增加。根据形成的复合物对电子转移和响应电流的阻碍,可以实现对凝血酶的免标记测定。实验结果表明,APTS-TMCS-MPS介孔孔道内的GNPs,可以提高孔道内电子的传递效率。在优化的实验条件下,该免标记DNA电化学传感器对凝血酶的检测线性范围是1.0×10-8~1.0×10-6 mol·L-1,检测限是7.5×10-9 mol·L-1(3σ)。     

Nano-Au/复合壳聚糖@纳米碳修饰金电极检测亚硝酸盐

该文介绍了以复合壳聚糖@纳米碳为基底固定纳米金,利用具有催化性的纳米碳及纳米金作为识别元素检测亚硝酸盐,从而制备了无生物物质标记且无电子媒介体的亚硝酸盐传感器。该修饰电极在亚硝酸盐浓度为8.0×10-6~2.0×10-4mol/L范围内有线性响应,线性相关系数r=0.9956。实验结果表明,该传感器具有制备简单、灵敏度高、稳定性好、线性范围宽等优点。     

Simple,fast, label-free,and nanoquencher-free system for operating multivalued DNA logic gates using polythymine templated CuNPs as signal reporters

Boolean logic devices play a key role in both traditional and nontraditional molecular logic circuits.This kind of binary logic,in which each bit is coded by (0,1),has only two output states—on or off (or high/low).Because of the finite computing capacity and variation,it is facing challenges from multivalued logic gates while processing high-density or uncertain/imprecise information.However,a low-cost,simple,and universal system that can perform different multivalued logic computations has not yet been developed,and remains a concept for further study.Herein,taking the ternary OR and INHIBIT logic gates as model devices,we present the fabrication of a novel simple,fast,label-free,and nanoquencher-free system for multivalued DNA logic gates using poly-thymine (T) templated copper nanoparticles (CuNPs) as signal reporters.The mixture of Cu2+ and ascorbic acid (AA) is taken as a universal platform for all ternary logic gates.Different kinds of poly-T strands and delicately designed complementary poly-adenine (A) strands are alternatively applied as ternary inputs to exhibit the ternary output states (low/0,medium/1,high/2).Notably,there are no nanoquenchers in this platform as poly-A strands can function as not only inputs but also efficient inhibitors of poly-T templated CuNPs.Moreover,all DNA are unlabeled single-strand DNA that do not need sophisticated labeling procedures or sequence design.The above design greatly reduces the operating time,costs,and complexity.More importantly,the ternary logic computations can be completed within 20 min because of the fast formation of CuNPs,and all of them share the same threshold values.     

Fluorescent Amino Acid Initiated de novo Cyclic Peptides for the Label-Free Assessment of Cell Permeability**

The major obstacle in applying peptides to intracellular targets is their low inherent cell permeability. Standard approaches to attach a fluorophore (e. g. FITC, TAMRA) can change the physicochemical properties of the parent peptide and influence their ability to penetrate and localize in cells. We report a label-free strategy for evaluating the cell permeability of cyclic peptide leads. Fluorescent tryptophan analogues 4-cyanotryptophan (4CNW) and β-(1-azulenyl)-L-alanine (AzAla) were incorporated into in vitro translated macrocyclic peptides by initiator reprogramming. We then demonstrate these efficient blue fluorescent emitters are good tools for monitoring peptide penetration into cells.     

Label-Free Study of the Global Cell Behavior during Exposure to Environmental Radiofrequency Fields in the Presence or Absence of Pro-Apoptotic or Pro-Autophagic Treatments

It remains controversial whether exposure to environmental radiofrequency signals (RF) impacts cell status or response to cellular stress such as apoptosis or autophagy. We used two label-free techniques, cellular impedancemetry and Digital Holographic Microscopy (DHM), to assess the overall cellular response during RF exposure alone, or during co-exposure to RF and chemical treatments known to induce either apoptosis or autophagy. Two human cell lines (SH-SY5Y and HCT116) and two cultures of primary rat cortex cells (astrocytes and co-culture of neurons and glial cells) were exposed to RF using an 1800 MHz carrier wave modulated with various environmental signals (GSM: Global System for Mobile Communications, 2G signal), UMTS (Universal Mobile Telecommunications System, 3G signal), LTE (Long-Term Evolution, 4G signal, and Wi-Fi) or unmodulated RF (continuous wave, CW). The specific absorption rates (S.A.R.) used were 1.5 and 6 W/kg during DHM experiments and ranged from 5 to 24 W/kg during the recording of cellular impedance. Cells were continuously exposed for three to five consecutive days while the temporal phenotypic signature of cells behavior was recorded at constant temperature. Statistical analysis of the results does not indicate that RF-EMF exposure impacted the global behavior of healthy, apoptotic, or autophagic cells, even at S.A.R. levels higher than the guidelines, provided that the temperature was kept constant.     

Rapid,Label-Free Prediction of Antibiotic Resistance in Salmonella typhimurium by Surface-Enhanced Raman Spectroscopy

The rapid identification of bacterial antibiotic susceptibility is pivotal to the rational administration of antibacterial drugs. In this study, cefotaxime (CTX)-derived resistance in Salmonella typhimurium (abbr. CTX^r-S. typhimurium) during 3 months of exposure was rapidly recorded using a portable Raman spectrometer. The molecular changes that occurred in the drug-resistant strains were sensitively monitored in whole cells by label-free surface-enhanced Raman scattering (SERS). Various degrees of resistant strains could be accurately discriminated by applying multivariate statistical analyses to bacterial SERS profiles. Minimum inhibitory concentration (MIC) values showed a positive linear correlation with the relative Raman intensities of I₉₉₀/I₁₃₄₈, and the R² reached 0.9962. The SERS results were consistent with the data obtained by MIC assays, mutant prevention concentration (MPC) determinations, and Kirby-Bauer antibiotic susceptibility tests (K-B tests). This preliminary proof-of-concept study indicates the high potential of the SERS method to supplement the time-consuming conventional method and help alleviate the challenges of antibiotic resistance in clinical therapy.     

Evaluation of kinetics using label-free optical biosensors

Optical biosensors provide a platform for qualitatively and quantitatively analyzing various biomolecular interactions. In addition to advantages such as label-free and high-throughput detection, these devices are also capable of measuring real-time binding curves in response to changes in optical properties of biomolecules. These kinetic data may be fitted to models to extract binding affinities such as association rates, dissociation rates, and equilibrium dissociation constants. In these biosensors, one of the binding pair is usually immobilized on a solid substrate for capturing the other. Due to the nature of these surface-based methods, mass transport effects and immobilization heterogeneity may cause problems when fitting the kinetic curves with the simple one-to-one Langmuir model. Here, real-time binding curves of various antibody–antigen reactions were obtained using an ellipsometry-based biosensor, and the results were fitted to the simple one-to-one model as well as a more sophisticated approach. The results show that the one-to-two model fitted much better to the curves than the one-to-one model. The two-site model may be explained by assuming two immobilization configurations on the surface. In summary, in fitting real-time curves obtained from optical biosensors, more sophisticated models are usually required to take surface-related issues, such as immobilization heterogeneity, and mass transport effects within targets, into account.     

Label-Free Quantitative Proteomics Reveals Differences in Molecular Mechanism of Atherosclerosis Related and Non-Related to Chronic Kidney Disease

The major cause of mortality in patients with chronic kidney disease (CKD) is atherosclerosis related to traditional and non-traditional risk factors. However, the understanding of the molecular specificity that distinguishes the risk factors for classical cardiovascular disease (CVD) and CKD-related atherosclerosis (CKD-A) is far from complete. In this study we investigated the disease-related differences in the proteomes of patients with atherosclerosis related and non-related to CKD. Plasma collected from patients in various stages of CKD, CVD patients without symptoms of kidney dysfunction, and healthy volunteers (HVs), were analyzed by a coupled label-free and mass spectrometry approach. Dysregulated proteins were confirmed by an enzyme-linked immunosorbent assay (ELISA). All proteomic data were correlated with kidney disease development and were subjected to bioinformatics analysis. One hundred sixty-two differentially expressed proteins were identified. By directly comparing the plasma proteomes from HVs, CKD, and CVD patients in one study, we demonstrated that proteins involved in inflammation, blood coagulation, oxidative stress, vascular damage, and calcification process exhibited greater alterations in patients with atherosclerosis related with CKD. These data indicate that the above nontraditional risk factors are strongly specific for CKD-A and appear to be less essential for the development of “classical” CVD.     

Novel portable electrical detection system for DNA SENSOR application

A novel, portable, electrical detection system was constructed for DNA sensor application to detect DNA hybridisation. The read-out circuit consists of an analogue circuit and a digital circuit. The analogue circuit with an IC MAX038 generates a sine wave with a constant frequency (10?kHz), which serves as the input for the DNA sensor. The DNA sensor consists of active and reference sensors. DNA hybridisation between the DNA probe and the target sequences causes changes in the conductance of the conductive membrane (DNA/MWCNTs) on the sensor surface, which lead to changes in the amplitude of the sine wave from the sensor output compared with that of the reference signal output (input sine wave). We used a digital circuit with a microprocessor (PIC33FJ256MC710) to determine the change in the amplitude of the sine wave signal of the sensor. From these digital data, the difference in the amplitudes of the active and reference sensors was calculated and displayed on the liquid crystal display. Measurement results show that the portable electrical detection system can detect DNA target concentrations as low as 0.16?µM. The detection of the amplified polymerase chain reaction sample and the reproducibility of the DNA sensor results were also determined using the designed readout circuit. The proposed electrical detection system is suitable for DNA sensor application.