基于CRISPR/Cas的分子诊断传感器在非核酸分子诊断中的应用

CRISPR/Cas不仅是一种重要的基因编辑工具,而且还是一种有效的分子诊断工具。目前基于CRISPR/Cas建立了一系列的分子诊断传感器系统,广泛应用于核酸、非核酸等检测过程中。与应用较广泛的核酸分子诊断传感器系统相比,基于CRISPR/Cas的非核酸检测系统目前尚未见系统性综述,因此本文围绕基于CRISPR/Cas12和CRISPR/Cas13建立的两大类非核酸分子传感器诊断系统的基本特征、工作流程及其检测原理等进行了全面综述,期望能为CRISPR/Cas分子诊断系统在体外诊断中的应用提供依据。     

Improved Detection of Magnetic Signals by a MEMS Sensor Using Stochastic Resonance

We introduce the behavior of the electrical output response of a magnetic field sensor based on microelectromechanical systems (MEMS) technology under different levels of controlled magnetic noise. We explored whether a particular level of magnetic noise applied on the vicinity of the MEMS sensor can improve the detection of subthreshold magnetic fields. We examined the increase in the signal-to-noise ratio (SNR) of such detected magnetic fields as a function of the magnetic noise intensity. The data disclosed an inverted U-like graph between the SNR and the applied magnetic noise. This finding shows that the application of an intermediate level of noise in the environment of a MEMS magnetic field sensor improves its detection capability of subthreshold signals via the stochastic resonance phenomenon.     

Continuous flow displacement immunosensors: a computational study

Numerical modeling has been used to investigate the disparity in performance and sensitivity that has been reported for flow displacement immunosensors based on bead-packed columns, membranes, and capillary tubes. The simulations strongly suggest that the high surface areas in the porous media systems may actually be detrimental to sensor performance because of large numbers of free antibody binding sites. Since the free antibody sites are created during the wash step in which the baseline is established, wash protocols are critical in optimizing the sensitivity for a given displacement sensor.     

Active Sensing System with In Situ Adjustable Sensor Morphology

Background

Despite the widespread use of sensors in engineering systems like robots and automation systems, the common paradigm is to have fixed sensor morphology tailored to fulfill a specific application. On the other hand, robotic systems are expected to operate in ever more uncertain environments. In order to cope with the challenge, it is worthy of note that biological systems show the importance of suitable sensor morphology and active sensing capability to handle different kinds of sensing tasks with particular requirements.

Methodology

This paper presents a robotics active sensing system which is able to adjust its sensor morphology in situ in order to sense different physical quantities with desirable sensing characteristics. The approach taken is to use thermoplastic adhesive material, i.e. Hot Melt Adhesive (HMA). It will be shown that the thermoplastic and thermoadhesive nature of HMA enables the system to repeatedly fabricate, attach and detach mechanical structures with a variety of shape and size to the robot end effector for sensing purposes. Via active sensing capability, the robotic system utilizes the structure to physically probe an unknown target object with suitable motion and transduce the arising physical stimuli into information usable by a camera as its only built-in sensor.

Conclusions/Significance

The efficacy of the proposed system is verified based on two results. Firstly, it is confirmed that suitable sensor morphology and active sensing capability enables the system to sense different physical quantities, i.e. softness and temperature, with desirable sensing characteristics. Secondly, given tasks of discriminating two visually indistinguishable objects with respect to softness and temperature, it is confirmed that the proposed robotic system is able to autonomously accomplish them. The way the results motivate new research directions which focus on in situ adjustment of sensor morphology will also be discussed.     

Multi-analyte surface plasmon resonance biosensing

Surface plasmon resonance (SPR) biosensors are affinity sensing devices exploiting a special mode of electromagnetic field-surface plasmon-polariton-to detect the binding of analyte molecules from a liquid sample to biomolecular recognition elements immobilized on the surface of the sensor. In this paper, we review advances of SPR biosensor technology towards detection systems for the simultaneous detection of multiple analytes (multi-analyte detection). In addition, we report application of a recently developed multichannel SPR sensor based on spectroscopy of surface plasmons and wavelength division multiplexing of sensing channels to multi-analyte detection.     

Novel system for real-time ex vivo lactate monitoring in human whole blood

The objective of the study was to evaluate the performance of an amperometric enzyme based lactate sensor and to investigate the possibility of replacing a double lumen catheter based blood withdrawal system with a heparin coated single lumen system. The inner lumen of a double lumen catheter which was placed in a peripheral vein was perfused with heparin solution. The outer lumen was used to collect heparinized blood samples at a defined flow rate. The single lumen system was attached to a heparinized catheter which was also placed in a peripheral vein. The undiluted blood samples were collected at a specified flow rate. A sensor flow chamber incorporating an amperometric thin-film lactate microbiosensor was placed in the sampling line for real-time lactate monitoring. Plasma lactate concentrations were measured during frequently performed hyperlactatemia bicycle ergometer experiments in six healthy volunteers (age 25.8±2.8 years, BMI 22.7±1 kg/m²). Additionally, plasma lactate was measured in real-time using the lactate sensors. The first three experiments were performed with a double lumen based catheter system whereas the following three experiments were performed with a heparin coated catheter system. The correlation coefficients of sensor readings and laboratory analyzer results in all six experiments were between 0.93 and 0.99, respectively (P<0.001). The miniaturized lactate sensors showed a linear range up to 25 mmol/l lactate concentration and 95% response times <30 s in undiluted serum. During the experiments maximum lactate concentrations of 14 mmol/l were achieved. Improvements of system performance using heparin coated catheter systems could be shown. The overall SD of the sensor readings compared to laboratory results using three double lumen catheter based systems was 0.91 mmol/l whereas the SD using three heparin coated systems was 0.65 mmol/l. In summary, real-time monitoring of lactate in human whole blood is feasible with such a device and can be improved by using heparin coated catheter systems.     

A whole-cell biosensor as in vitro alternative to skin irritation tests

This study presents the time-resolved detection of chemically induced stress upon intracellular signaling cascades by using genetically modified sensor cells based on the human keratinocyte cell line HaCaT. The cells were stably transfected with a HSP72-GFP reporter gene construct to create an optical sensor cell line expressing a stress-inducible reporter protein. The time- and dose-dependent performance of the sensor cells is demonstrated and discussed in comparison to a label-free impedimetric monitoring approach (electric cell-substrate impedance sensing, ECIS). Moreover, a microfluidic platform was established based on μSlidesI(0,4)Luer to allow for a convenient, sterile and incubator-independent time-lapse microscopic observation of the sensor cells. Cell growth was successfully achieved in this microfluidic setup and the cellular response to a cytotoxic substance could be followed in real-time and in a non-invasive, sensitive manner. This study paves the way for the development of micro-total analysis systems that combine optical and impedimetric readouts to enable an overall quantitative characterization of changes in cell metabolism and morphology as a response to toxin exposure. By recording multiple parameters, a detailed discrimination between competing stress- or growth-related mechanisms is possible, thereby presenting an entirely new in vitro alternative to skin irritation tests.     

Fibre-optic biosensor based on luminescence and immobilized enzymes: Microdetermination of sorbitol,ethanol and oxaloacetate

We have investigated highly selective and ultrasensitive biosensors based on luminescent enzyme systems linked to optical transducers. A fibre-optic sensor with immobilized enzymes was designed; the solid-phase bioreagent was maintained in close contact with the tip of a glass fibre bundle connected to the photomultiplier tube of a luminometer. A bacterial luminescence fibre-optic sensor was used for the microdetermination of NADH. Various NAD(P)-dependent enzymes, sorbitol dehydrogenase, alcohol dehydrogenase and malate dehydrogenase, were co-immobilized on preactivated polyamide membranes with the bacterial system and used for the microdetermination of sorbitol, ethanol and oxaloacetate at the nanomolar level with a good precision.     

Identification of a single genome by electron paramagnetic resonance (EPR) with nitroxide-labeled oligonucleotide probes

Nitroxide-labeled nucleic acids are used as a molecular size sensor to identify as few as one genome under polymerase chain reaction (PCR) conditions by electron paramagnetic resonance (EPR) spectroscopy. DNA identification is based on differences in the EPR spectra of mono-nitroxide-labeled nucleic acids. The experimental data imply that rapid DNA identification can be achieved in many systems by EPR at the molecular level.     

Catecholamine detection using enzymatic amplification

Different amplification sensors based on the substrate recycling principle were investigated with respect to their applicability to catecholamine detection. In the bioelectrocatalytic approach, glassy carbon electrodes were modified by laccase or a PQQ-dependent glucose dehydrogenase. Substrate recycling occurs and the detection limit is in the lower nanomolar concentration range (e.g. 10 nM dopamine and 1 nM noradrenaline for the laccase- and glucose dehydrogenase-modified electrodes, respectively). Combinations of glucose dehydrogenase with laccase or tyrosinase were investigated as bienzymatic probes. Among the systems we studied, the laccase/glucose dehydrogenase sensor is the most sensitive (detection limit: 0·5 nM adrenaline). The selectivities of the different sensor systems are discussed. Application of the laccase/glucose dehydrogenase electrode in different media (i.e. brain homogenate, heart effluate) was successfully shown. For samples with high concentrations of interfering substances (uric and ascorbic acid), the interferences can be effectively removed using enzymatic methods.     

NMR Spectra of Some Monosaccharides of Galactopyranose Series in Deuterium Oxide

Application of a hot-wire viscosity sensor, which measures the intensity of the natural convection around a hot wire, have been limited to the stagnant systems. For the adaptation of the hot-wire viscosity sensor to flowing systems, the hot-wire viscosity sensor was protected from the effects of the flow by using a stainless steel shield. The shield was normally left open, and was closed only when the measurement was done. Although the hot-wire sensor response was influenced by the flow outside the shield through the high thermal conductivity of the shield material, this effect could be eliminated when the sensor was used at a sufficiently high flow rate outside the shield. The shielded hot-wire sensor responded well to changes in the viscosity of the test fluid in a wide range of viscosity. The effects of the size of the shield and the heat-flux applied on the sensor response were investigated. The shielded hot-wire viscosity sensor was used for the monitoring of the hydrolysis of starch by α-amylase.     

Comparative analysis of prototype two-component systems with either bifunctional or monofunctional sensors: differences in molecular structure and physiological function

Signal transduction by a traditional two-component system involves a sensor protein that recognizes a physiological signal, autophosphorylates and transfers its phosphate, and a response regulator protein that receives the phosphate, alters its affinity toward specific target proteins or DNA sequences and causes change in metabolic activity or gene expression. In some cases the sensor protein, when unphosphorylated, has a positive effect upon the rate of dephosphorylation of the regulator protein (bifunctional sensor), whereas in other cases it has no such effect (monofunctional sensor). In this work we identify structural and functional differences between these two designs. In the first part of the paper we use sequence data for two-component systems from several organisms and homology modelling techniques to determine structural features for response regulators and for sensors. Our results indicate that each type of reference sensor (bifunctional and monofunctional) has a distinctive structural feature, which we use to make predictions regarding the functionality of other sensors. In the second part of the paper we use mathematical models to analyse and compare the physiological function of systems that differ in the type of sensor and are otherwise equivalent. Our results show that a bifunctional sensor is better than a monofunctional sensor both at amplifying changes in the phosphorylation level of the regulator caused by signals from the sensor and at attenuating changes caused by signals from small phosphodonors. Cross-talk to or from other two-component systems is better suppressed if the transmitting sensor is monofunctional, which is the more appropriate design when such cross-talk represents pathological noise. Cross-talk to or from other two-component systems is better amplified if the transmitting sensor is bifunctional, which is the more appropriate design when such cross-talk represents a physiological signal. These results provide a functional rationale for the selection of each design that is consistent with available experimental evidence for several two-component systems.     

Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation

There is a groundswell of interest in using genetically engineered sensor bacteria to study gut microbiota pathways, and diagnose or treat associated diseases. Here, we computationally identify the first biological thiosulfate sensor and an improved tetrathionate sensor, both two‐component systems from marine Shewanella species, and validate them in laboratory Escherichia coli. Then, we port these sensors into a gut‐adapted probiotic E. coli strain, and develop a method based upon oral gavage and flow cytometry of colon and fecal samples to demonstrate that colon inflammation (colitis) activates the thiosulfate sensor in mice harboring native gut microbiota. Our thiosulfate sensor may have applications in bacterial diagnostics or therapeutics. Finally, our approach can be replicated for a wide range of bacterial sensors and should thus enable a new class of minimally invasive studies of gut microbiota pathways.     

Phosphorescent porphyrin probes in biosensors and sensitive bioassays

Platinum(II) and palladium(II) complexes of porphyrins and related tetrapyrrolic pigments emit strong phosphorescence at room temperatures, which is characterized by long lifetimes falling into the sub-millisecond range and long-wave spectral characteristics. These features make the dyes useful as probes for a number of bioanalytical applications, particularly those employing time-resolved fluorescent detection. They can provide high sensitivity and selectivity, together with rather simple instrumental set-up. A number of analytical systems are now under development that are based on the use of phosphorescent porphyrin probes. Experimental results are presented on the following systems: (i) fibre-optic phosphorescence lifetime-based oxygen sensor on the basis of hydrophobic platinum-porphyrins and development of advanced sensing materials and prototype instrumentation; (ii) practical applications of the optical oxygen sensor, including a sensitive immunosensor that employs glucose oxidase labels, a rapid screening method for cell viability in microtitre-plate format, non-destructive measurement of oxygen in packaged foods and reagentless biosensors for metabolites (glucose, lactate); and (iii) the use of water-soluble platinum- and palladium-porphyrins as labels for ultra-sensitive time-resolved phosphorescence immunoassays.     

生物发光及化学发光在生物医学领域中应用的进展

生物发光和化学发光在生物医学领域内的应用主要包括细胞学检测,分子生物学、卫生学检测,生物传感器、脂质过氧化检测和药物筛选等六个方面,其中细胞学检测主要是利用细胞内ＡＴＰ导致的虫荧光素酶发光进行活细胞计数,目前已实现快速、动态、单细胞分析;同时发现了一些新的与生物或化学发光有关的细胞学指标。分子生物学领域内的应用主要为报告基因和分子杂交,近年来又有人推出了生物发光实时ＤＮＡ测序技术。卫生学检测则主要     

Potential errors in conventional DOT measurement techniques in shake flasks and verification using a rotating flexitube optical sensor

Background  

Dissolved oxygen tension (DOT) is an important parameter for evaluating a bioprocess. Conventional means to measure DOT in shake flasks using fixed Clark-type electrodes immersed in the bulk liquid are problematic, because they inherently alter the hydrodynamics of the systems. Other approaches to measure DOT that apply fluorescing sensor spots fixed at the inside wall of a shake flask are also suboptimal. At low filling volumes for cultivating microorganisms with a high oxygen demand, the measured DOT signal may be erroneous. Here, the sensor spot is sometimes exposed to gas in the head space of the flask. Merely repositioning the sensor spot elsewhere in the flask does not address this problem, since there is no location in the shake flask that is always covered by the rotating bulk liquid. Thus, the aim of this prospective study is first, to verify the systemic error of Clark-type electrodes for measuring DOT in shake flasks. The second principle aim is to use the newly built "flexitube optical sensor" to verify potential errors in conventional optical DOT measurements based on fixed sensor spots.     

Protein phosphorylation in chemotaxis and two-component regulatory systems of bacteria

Two-component regulatory systems appear to be widespread in bacteria. Phosphorylation has been demonstrated in three of the known systems and correlated with in vivo function in two cases (Che and Ntr). Although phosphorylation of sensor and regulator proteins has so far been observed exclusively in vitro, transient protein phosphorylation could provide a basis for the mechanism of signal transduction in these bacterial systems. There is currently insufficient evidence, however, to establish the precise functional relationship(s) between the conserved sensor and regulator sequences, phosphorylation, and the detailed mechanism involved in signal transduction via the sensor and regulator proteins.     

Localization algorithm in wireless sensor networks based on semi-supervised manifold learning and its application

Localization of mobile nodes in wireless sensor network gets more and more important, because many applications need to locate the source of incoming measurements as precise as possible. Many previous approaches to the location-estimation problem need know the theories and experiential signal propagation model and collect a large number of labeled samples. So, these approaches are coarse localization because of the inaccurate model, and to obtain such data requires great effort. In this paper, a semi-supervised manifold learning is used to estimate the locations of mobile nodes in a wireless sensor network. The algorithm is used to compute a subspace mapping function between the signal space and the physical space by using a small amount of labeled data and a large amount of unlabeled data. This mapping function can be used online to determine the location of mobile nodes in a sensor network based on the signals received. We use independent development nodes to setup the network in metallurgical industry environment, outdoor and indoor. Experimental results show that we can achieve a higher accuracy with much less calibration effort as compared with RADAR localization systems.     

Multimedia information retrieval and environmental monitoring: Shared perspectives on data fusion

Computer-based remote monitoring of our environment is increasingly based on combining data derived from in-situ-sensors with data derived from remote sources, such as satellite images or CCTV. In such deployments it is necessary to continuously monitor the accuracy of each of the sensor data streams so that we can account for sudden failures of sensors, or errors due to calibration drive or biofouling. In multimedia information retrieval (MMIR), we search through archives of multimedia artefacts like video programs, by implementing several independent retrieval systems or agents, and we combine the outputs of each retrieval agent in order to generate an overall ranking. In this paper we draw parallels between these seemingly very different applications and show how they share several similarities. In the case of environmental monitoring we also need some mechanism by which we can establish the trust and reputation of each contributing sensor, though this is something we do not need in MMIR. In this paper we present an outline of a trust and reputation framework we have developed and deployed for monitoring the performance of sensors in a heterogeneous sensor network.