细菌群体感应信号分子的光电检测技术

群体感应（quorum sensing，QS）是一种依赖菌群密度的细菌交流系统。在探究细菌群体感应系统的调控机制中，对QS信号分子的鉴别和检测是不可或缺的环节，其对生命科学、药学等领域涉及细菌等微生物的相互作用、高效检测和作用机制解析等具有重要的参考意义。本文在总结不同类型细菌QS信号分子来源和结构的基础上，对QS信号分子的光电检测方法和技术进行了综述，重点对光电传感检测的敏感介质、传感界面、传感机制及测试效果进行探讨，同时关注了将微流控芯片分析技术应用于细菌QS信号分子原位监测的相关研究进展。     

Polycation-assisted DNA detection by reduction triggered fluorescence amplification probe

We have developed a fluorescence detection system for DNA, assisted by a comb-type cationic polymer (PLL-g-DX), for accelerating the reaction turnover. The combination of fluorogenic DNA probes with a comb-type cationic polymer has been demonstrated to be an effective means of signal amplification during the detection process. The method described herein represents a simple and enzyme-free detection.     

基于谱域相干的细胞动态检测技术

将谱域光学相干层析技术（Spectral-domain Optical Coherence Tomography,SD-OCT）应用于细胞膜厚的动态检测,引入了光谱干涉测量方法,通过对干涉光谱信号进行离散傅里叶变换得到表示物体深度位置信息的光程差信号。搭建了一套SD-OCT测量系统并设计了专门用于细胞检测的样品平台,检测精度达到μm量级,信噪比达70 db,是一种快速、实时、非接触的细胞分子膜厚动态检测技术。     

Signal detection theory,detectability and stochastic resonance effects

 Stochastic resonance is a phenomenon in which the performance of certain non-linear detectors can be enhanced by the addition of appropriate levels of random noise. Signal detection theory offers a powerful tool for analysing this type of system, through an ability to separate detection processes into reception and classification, with the former generally being linear and the latter always non-linear. Through appropriate measures of signal detectability it is possible to decide whether a local improvement in detection via stochastic resonance occurs due to the non-linear effects of the classification process. In this case, improvement of detection through the addition of noise can never improve detection beyond that of a corresponding adaptive system. Signal detection and stochastic resonance is investigated in several integrate-and-fire neuron models. It is demonstrated that the stochastic resonance observed in spiking models is caused by non-linear properties of the spike-generation process itself. The true detectability of the signal, as seen by the receiver part of the spiking neuron (the integrator part), decreases monotonically with input noise level for all signal and noise intensities. Received: 3 April 2001 / Accepted in revised form: 8 March 2002     

Digital Imaging as a Detection Method for a Fluorescent Protease Assay in 96-Well and Miniaturized Assay Plate Formats

The demand to increase throughput in HTS programs, without a concomitant addition to costs, has grown significantly during the past few years. One approach to handle this demand is assay miniaturization, which can provide greater throughput, as well as significant cost savings through reduced reagent costs. Currently, one of the major challenges facing assay miniaturization is the ability to detect the assay signal accurately and rapidly in miniaturized formats. Digital imaging is a detection method that can measure fluorescent or luminescent signals in these miniaturized formats. In this study, an imaging system capable of detecting the signal from a fluorescent protease assay in multiple plate formats was used to evaluate this detection method in an HTS environment. A direct comparison was made between the results obtained from the imaging system and a fluorescent plate reader by screening 8,800 compounds in a 96-well plate format. The imaging system generated similar changes in relative signal for each well in the screen, identified the same active compounds, and yielded similar IC(50) values as compared to the plate reader. When a standard protease inhibitor was evaluated in 96-, 384-, 864-, and 1536-well plates using imaging detection, similar IC(50) values were obtained. Furthermore, similar dose-response curves were generated for the compound in 96- and 384-well assay plates read in a plate reader. These results provide support for digital imaging as an accurate and rapid detection method for high-density microtiter plates.     

A Deep Learning Architecture for P300 Detection with Brain-Computer Interface Application

In this paper, a brain-computer interface (BCI) system for character recognition is proposed based on the P300 signal. A P300 speller is used to spell the word or character without any muscle movement. P300 detection is the first step to detect the character from the electroencephalogram (EEG) signal. The character is recognized from the detected P300 signal. In this paper, sparse autoencoder (SAE) and stacked sparse autoencoder (SSAE) based feature extraction methods are proposed for P300 detection. This work also proposes a fusion of deep-features with the temporal features for P300 detection. A SSAE technique extracts high-level information about input data. The combination of SSAE features with the temporal features provides abstract and temporal information about the signal. An ensemble of weighted artificial neural network (EWANN) is proposed for P300 detection to minimize the variation among different classifiers. To provide more importance to the good classifier for final classification, a higher weightage is assigned to the better performing classifier. These weights are calculated from the cross-validation test. The model is tested on two different publicly available datasets, and the proposed method provides better or comparable character recognition performance than the state-of-the-art methods.     

Detection of tripping gait patterns in the elderly using autoregressive features and support vector machines

Elderly tripping falls cost billions annually in medical funds and result in high mortality rates often perpetrated by pulmonary embolism (internal bleeding) and infected fractures that do not heal well. In this paper, we propose an intelligent gait detection system (AR-SVM) for screening elderly individuals at risk of suffering tripping falls. The motivation of this system is to provide early detection of elderly gait reminiscent of tripping characteristics so that preventive measures could be administered. Our system is composed of two stages, a predictor model estimated by an autoregressive (AR) process and a support vector machine (SVM) classifier. The system input is a digital signal constructed from consecutive measurements of minimum toe clearance (MTC) representative of steady-state walking. The AR-SVM system was tested on 23 individuals (13 healthy and 10 having suffered at least one tripping fall in the past year) who each completed a minimum of 10 min of walking on a treadmill at a self-selected pace. In the first stage, a fourth order AR model required at least 64 MTC values to correctly detect all fallers and non-fallers. Detection was further improved to less than 1 min of walking when the model coefficients were used as input features to the SVM classifier. The system achieved a detection accuracy of 95.65% with the leave one out method using only 16 MTC samples, but was reduced to 69.57% when eight MTC samples were used. These results demonstrate a fast and efficient system requiring a small number of strides and only MTC measurements for accurate detection of tripping gait characteristics.     

Stochastic resonance is applied to quantitative analysis for weak chromatographic signal of roxithromycin in beagle dog plasma

Based on the theory of stochastic resonance, the signal to noise ratio (SNR) of HPLC/UV chromatographic signal of roxithromycin is enhanced by cooperation of signal, noise and nonlinear system. A simple new method for the determination of low concentration of roxithromycin in beagle dog plasma is presented. Using signal enhancement by stochastic resonance, this method extends the limit of quantitation from the reported 0.5 to 0.1 microg/ml. During validation of the new method, HPLC/MS was used as a comparison technique. The results indicate that the recovery and low concentrations of roxithromycin in beagle dog plasma were equivalent between the two methods (P>0.05). Stochastic resonance may be a promising tool for improving detection limits in trace analysis.     

A novel microfluidic biosensor based on an electrical detection system for alpha-fetoprotein

Conventional immunoassays are labor intensive, expensive and time consuming and require large pieces of equipment for detection. Therefore, we have developed and characterized a novel immunoassay methodology comprised of microbeads and microbiochips. In this method, microbeads are used to filter and immobilize antibodies and an immuno-gold silver staining (IGSS) method is then used to amplify electrical signals that correspond to the bound antibodies. The chip used for this system is composed of an inexpensive and biocompatible polydimethylsiloxane (PDMS) layer over a Pyrex glass substrate that contains a platinum (Pt) microelectrode, which is used to detect the electrical signal in this system, the microelectrode is fabricated on the substrate and a microchannel and pillar-type microfilter is formed in the PDMS layer. A sandwich immunoassay approach was applied to detect alpha-fetoprotein (AFP), a cancer biomarker, using this system. The results of this study showed that the time required for a complete assay was reduced by 1h and a detection limit as low as 1 ng/mL was attained when this system used, which indicates that similar bead-based electrical detection systems could be used for the diagnosis of many forms of cancer.     

Dual signal amplification for highly sensitive electrochemical detection of uropathogens via enzyme-based catalytic target recycling

We report an ultrasensitive electrochemical approach for the detection of uropathogen sequence-specific DNA target. The sensing strategy involves a dual signal amplification process, which combines the signal enhancement by the enzymatic target recycling technique with the sensitivity improvement by the quantum dot (QD) layer-by-layer (LBL) assembled labels. The enzyme-based catalytic target DNA recycling process results in the use of each target DNA sequence for multiple times and leads to direct amplification of the analytical signal. Moreover, the LBL assembled QD labels can further enhance the sensitivity of the sensing system. The coupling of these two effective signal amplification strategies thus leads to low femtomolar (5fM) detection of the target DNA sequences. The proposed strategy also shows excellent discrimination between the target DNA and the single-base mismatch sequences. The advantageous intrinsic sequence-independent property of exonuclease III over other sequence-dependent enzymes makes our new dual signal amplification system a general sensing platform for monitoring ultralow level of various types of target DNA sequences.     

Novel signal amplification technology with applications in DNA and protein detection systems.

A non-enzymatic approach to signal amplification has practical advantages over conventional target amplification methods. We have designed a simple, cost-efficient signal amplification system that can be used to enhance the detection of nucleic acids or protein. The signal amplification process requires initial capture of analyte by a specific probe, which, depending on the analyte, can be an oligomer or an antibody. Once the analyte is captured, amplification moieties are applied to significantly enhance the sensitivity of analyte detection. Nucleic acid amplification is typically greater than 1000-fold, increasing the sensitivity of target detection to less than 1 amol/100 microL. This amplification strategy presents a very flexible system with components that are easily altered to accommodate diverse assay requirements.     

Ozone-3,6-dihydroxynaphtha-2,7-disulphonate chemiluminescence system is used for online ozone detection

The chemiluminescence (CL) reaction between ozone and 3,6-dihydroxynaphtha-2,7-disulphonate (DNDS) was found under alkaline conditions. Therefore, a novel CL system for ozone detection was established. The CL signal of the CL system is weak, and the CL signal is enhanced by adding nonionic surfactants. It was found that adding 16.4 g/l Triton X-100 can enhance the CL signal. The CL reagent activated by ultraviolet (UV) light produced a CL signal was nearly 10 times stronger than the CL reagent not activated by UV light; the CL signal was enhanced by adding 8 g/l NaHCO₃ to the CL reagent irradiated by UV light. Through the optimization of these test conditions, a high-selectivity, high-sensitivity online detection method for ozone CL was established. The linear range was 0.5–150 ppbv, and the limit of detection (LOD) was 0.092 ppbv (S/N = 3).     

Microarray gene expression analysis free of reverse transcription and dye labeling

A new microarray system has been developed for gene expression analysis using cationic gold nanoparticles with diameters of 250 nm as a target detection reagent. The approach utilizes nonlabeled target molecules hybridizing with complementary probes on the array, followed by incubation in a colloidal gold solution. The hybridization signal results from the precipitation of nanogold particles on the hybridized spots due to the electrostatic attraction of the cationic gold particles and the anionic phosphate groups in the target DNA backbone. In contrast to conventional fluorescent detection, this nanoparticle-based detection system eliminates the target labeling procedure. The visualization of hybridization signals can be accomplished with a flatbed scanner instead of a confocal laser scanner, which greatly simplifies the process and reduces the cost. The sensitivity is estimated to be less than 2 pg of DNA molecules captured on the array surface. The signal from hybridized spots quantitatively represents the amount of captured target DNA and therefore permits quantitative gene expression analysis. Cross-array reproducibility is adequate for detecting twofold or less signal changes across two microarray experiments.     

Signal production and detection specificity in Vibrio CqsA/CqsS quorum-sensing systems

Quorum sensing is a process of bacterial cell-cell communication that enables populations of cells to carry out behaviours in unison. Quorum sensing involves detection of the density-dependent accumulation of extracellular signal molecules called autoinducers that elicit population-wide changes in gene expression. In Vibrio species, CqsS is a membrane-bound histidine kinase that acts as the receptor for the CAI-1 autoinducer which is produced by the CqsA synthase. In Vibrio cholerae, CAI-1 is (S)-3-hydroxytridecan-4-one. The C170 residue of V. cholerae CqsS specifies a preference for a ligand with a 10-carbon tail length. However, a phenylalanine is present at this position in Vibrio harveyi CqsS and other homologues, suggesting that a shorter CAI-1-like molecule functions as the signal. To investigate this, we purified the V. harveyi CqsS ligand, and determined that it is (Z)-3-aminoundec-2-en-4-one (Ea-C8-CAI-1) carrying an 8-carbon tail. The V. harveyi CqsA/CqsS system is exquisitely selective for production and detection of this ligand, while the V. cholerae CqsA/CqsS counterparts show relaxed specificity in both production and detection. We isolated CqsS mutants in each species that display reversed specificity for ligands. Our analysis provides insight into how fidelity is maintained in signal transduction systems.     

Biosensor for rapid phosphate monitoring in a sequencing batch reactor (SBR) system

A thick-film phosphate biosensor based on hydrogel immobilized pyruvate oxidase (POD) has been developed for rapid phosphate process control monitoring in an experimental sequencing batch reactor (SBR) system. We have employed a phosphate biosensor in an off-line monitoring of phosphate concentrations in a bench scale SBR. Measurements with biosensor show a good correlation (r2=0.98) with those of commercial colorimetric phosphate testing kits. The signal response time was 1 min with a detection limit of 5 microM. The biosensor method showed a good operational stability, needed less experimental procedures and a small sample size (approximately 20 microl). This allows its practical application for rapid phosphate measurements to obtain real time process data in a SBR system.     

An Improved Nonfluorescent Detection System for in Situ Hybridization in Plants

Molecular cytogenetics, particularly the localization of DNA sequences by in situ hybridization, has increased our understanding about the genomic structure of plants and animals. We demonstrate here the application of an improved nonfluorescent in situ hybridization system detection (DAKO GenPoint system) to plant chromosomes. Using this system, highly repetitive 18S-25S rRNA genes were mapped on Vicia faba chromosomes (2n = 12). The modified method of this horseradish peroxidase based enzymatic detection system gave satisfactory results that are comparable to fluorescent signal detection.     

一种细胞外囊泡的流式检测方案的探索与建立

该研究旨在探索细胞外囊泡(extracellular vesicles,EVs)分析的标准化流程,建立一种高效的EVs检测方法,为EVs功能及临床转化研究提供技术支撑.实验采用经典的超速离心法分离EVs,借助已知直径的聚苯乙烯微球设定并优化流式检测EVs的参数,联合应用散色光信号及荧光信号对EVs进行双参数分析鉴定,最...     

Signal localization: a new approach in signal discovery

A new approach for statistical association signal identification is developed in this paper. We consider a strategy for nonprecise signal identification by extending the well‐known signal detection and signal identification methods applicable to the multiple testing problem. Collection of statistical instruments under the presented approach is much broader than under the traditional signal identification methods, allowing more efficient signal discovery. Further assessments of maximal value and average statistics in signal discovery are improved. While our method does not attempt to detect individual predictors, it instead detects sets of predictors that are jointly associated with the outcome. Therefore, an important application would be in genome wide association study (GWAS), where it can be used to detect genes which influence the phenotype but do not contain any individually significant single nucleotide polymorphism (SNP). We compare power of the signal identification method based on extremes of single p‐values with the signal localization method based on average statistics for logarithms of p‐values. A simulation analysis informs the application of signal localization using the average statistics for wide signals discovery in Gaussian white noise process. We apply average statistics and the localization method to GWAS to discover better gene influences of regulating loci in a Chinese cohort developed for risk of nasopharyngeal carcinoma (NPC).     

Interference with Saville's method in determination of low-molecular weight S-nitrosothiols by ultrafiltration.

To detect low-molecular weight S-nitrosothiols in human plasma, we used a system combining HPLC for separation and Saville's method for colorimetric detection of S-nitrosothiols. The sensitivity and detection limit was 1-2 nM for both S-nitrosocysteine and S-nitrosoglutathione. When plasma was analyzed after ultrafiltration (with units requiring higher g force [5000 g], irrespective to the material of the membrane) to eliminate high molecular substances, a signal corresponding to S-nitrosoglutahione was recognized. This signal behaved as real S-nitrosoglutathione as it was partially Hg(2+)-sensitive and gradually decayed with time. However, the use of pre-washed units or another ultrafiltration unit that required lower g force (1800 g) or direct application of plasma to the HPLC-Saville's method system did not result in such signal. Based on these observations, it is important to be aware of the interference originating from the ultrafiltration unit and its potential effect on the precise quantification of low molecular weight S-nitrosothiols using Saville's method.     

Homogeneous amplified single-molecule detection: Characterization of key parameters

We recently presented a method that enables single-molecule enumeration by transforming specific molecular recognition events at nanometer dimensions to micrometer-sized DNA macromolecules. This transformation process is mediated by target-specific padlock probe ligation, followed by rolling circle amplification (RCA), resulting in the creation of one rolling circle product (RCP) for each recognized target. The transformation makes optical detection and quantification possible using standard fluorescence microscopy by counting the number of generated RCPs in a sample pumped through a microfluidic channel. In this study, we demonstrate that confocal volume definition is crucial to achieve high-precision measurements in the microfluidic quantification (coefficient of variance typically 3%). We further demonstrate that complementary sequence motifs between RCPs is only a weak inducer of aggregates and that all detection sites of the RCPs are occupied at detection oligonucleotide concentrations greater than 5 nM if hybridized in the proper buffer conditions. Therefore, the signal/noise ratio is limited by the number of detection sites. By increasing the density of detection sites in the RCP by a factor of 1.9, we show that the optical signal/noise level can be increased from 42 to 75.