One-step homogeneous protein detection based on aptamer probe and fluorescence cross-correlation spectroscopy

A new protein assay based on fluorescence cross-correlation spectroscopy (FCCS) and aptamer probe is developed. In this assay, two spectrally distinct fluorophores labeled aptamer probes are used to recognize and detect thrombin through a sandwich reaction. The sandwich complexes are diffused through a confocal detection volume. The cross-correlation signals can be observed only at the presence of the aptamer probes-protein sandwich complexes. Thrombin is selected as a target to validate the assay. The whole detection process can be completed within an hour with low-nanomolar sensitivity and high specificity. The novel aptamer-based FCCS detection offers a simple, rapid and sensitive method for protein analysis in a homogeneous format.     

General colorimetric detection of proteins and small molecules based on cyclic enzymatic signal amplification and hairpin aptamer probe

In this work, we developed a simple and general method for highly sensitive detection of proteins and small molecules based on cyclic enzymatic signal amplification (CESA) and hairpin aptamer probe. Our detection system consists of a hairpin aptamer probe, a linker DNA, two sets of DNA-modified AuNPs, and nicking endonuclease (NEase). In the absence of a target, the hairpin aptamer probe and linker DNA can stably coexist in solution. Then, the linker DNA can assemble two sets of DNA-modified AuNPs, inducing the aggregation of AuNPs. However, in the presence of a target, the hairpin structure of aptamer probe is opened upon interaction with the target to form an aptamer probe-target complex. Then, the probe-target complex can hybridize to the linker DNA. Upon formation of the duplex, the NEase recognizes specific nucleotide sequence and cleaves the linker DNA into two fragments. After nicking, the released probe-target complex can hybridize with another intact linker DNA and the cycle starts anew. The cleaved fragments of linker DNA are not able to assemble two sets of DNA-modified AuNPs, thus a red color of separated AuNPs can be observed. Taking advantage of the AuNPs-based sensing technique, we are able to assay the target simply by UV-vis spectroscopy and even by the naked eye. Herein, we can detect the human thrombin with a detection limit of 50 pM and adenosine triphosphate (ATP) with a detection limit of 100 nM by the naked eye. This sensitivity is about 3 orders of magnitude higher than that of traditional AuNPs-based methods without amplification. In addition, this method is general since there is no requirement of the NEase recognition site in the aptamer sequence. Furthermore, we proved that the proposed method is capable of detecting the target in complicated biological samples.     

Label-free electrochemical detection of protein based on a ferrocene-bearing cationic polythiophene and aptamer

Two label-free electrochemical methods for the detection of human alpha-thrombin using a water-soluble, ferrocene-functionalized polythiophene transducer and a single-stranded oligonucleotide aptamer probe are described. The first approach is a direct method in which the recorded current decreases upon addition of the targeted protein. The second one requires more steps and the additional utilization of PNA probes and nuclease enzyme. This indirect method leads to an increase of the electrical signal as a function of the concentration of human alpha-thrombin with a detection limit of 75 fmol.     

Electrochemical biosensor for detection of adenosine based on structure-switching aptamer and amplification with reporter probe DNA modified Au nanoparticles

In the present study, an electrochemical sensing strategy for highly sensitive detection of small molecules was developed based on switching structures of aptamers from DNA/DNA duplex to DNA/target complex. A gold electrode was first modified with gold nanoparticles (AuNPs), and thiolated capture probe was immobilized onto the electrode via sulfur-gold affinity. Then, a "sandwich-type" strategy was employed, which involved a linker DNA containing antiadenosine aptamer sequence and reporter DNA loaded on AuNPs. In the presence of adenosine, the aptamer part bound with adenosine and folded to the complex structure. As a result, the reporter probes together with AuNPs were released into solution and reduced a decrease in peak current. With the enhancement effect of AuNPs, a detection limit as low as 1.8 x 10(-10) M for adenosine was achieved. The sensor exhibited excellent selectivity against other nucleosides and could be used to detect adenosine from real human serum samples.     

Sub-femtomolar electrochemical detection of DNA hybridization based on latex/gold nanoparticle-assisted signal amplification

We report a relatively simple electrostatic method for modifying submicrometer-size latex spheres with gold nanoparticles (AuNPs) based on layer-by-layer modification of the latex by polyelectrolytes. The AuNP coverages for 343- and 501-nm-diameter spheres were 4.0 x 10 (10) +/- 1.3 x 10 (10) and 8.2 x 10 (10) +/- 2.7 x 10 (10) particles cm (-2), respectively, which is an increase of 1 order of magnitude on the previously reported coverage at latex-AuNPs using streptavidin-biotin binding (Kawde, A.N.; Wang, J. Electroanalysis 2004, 16, 101-107). Due to the fact that the AuNPs used here are also of a larger size (mean diameter 15.5 +/- 1.6 nm, cf. 5 nm), this represents an increase of 2 orders of magnitude in the number of Au atoms delivered per sphere. The spheres were attached to DNA probes specific to E. coli and used to detect probe hybridization by dissolution of the AuNPs, followed by measurement of Au (3+) ions by anodic stripping voltammetry (ASV). Use of differential pulse voltammetry for the stripping step, along with optimization of the ASV conditions, enabled a detection limit of 0.5 fM, which is, to the best of our knowledge, equal or lower than previous voltammetric nanoparticle methods for detection of DNA hybridization.     

Highly sensitive and selective bifunctional oligonucleotide probe for homogeneous parallel fluorescence detection of protein and nucleotide sequence

The existing isothermal polymerization-based signal amplification assays are usually accomplished via two strategies: rolling circle amplification (RCA) and circular strand-displacement polymerization. In essence, the two techniques are based on cyclical nucleic acid strand-displacement polymerization (CNDP), limiting the application of isothermal polymerization in medical diagnosis and bioanalysis. In the present study, circular common target molecule (non-nucleic acid strand)-displacement polymerization (CCDP) is developed to amplify the fluorescence signal for biomolecule assays, extending isothermal polymerization to an aptameric system without any medium. Via combining an aptamer with a common hairpin DNA probe, we designed a self-blocked fluorescent bifunctional oligonucleotide probe (signaling probe) for the homogeneous parallel detection of two disease markers, PDGF-BB and the p53 gene. On the basis of CNDP and CCDP signal amplification, highly sensitive (e.g., detecting PDGF down to the concentration level of 1.8 × 10(-10) M) and selective detection (no interference even in the presence of a significantly higher concentration (7-200 times) of nontarget proteins) was accomplished, and the linear response range was considerably widened. Furthermore, the bifunctional signaling probe exhibits impressive simplicity, convenience, and short detection time. Herein, the design of the signaling probe was described, factors influencing fluorescence signal were investigated, analytical properties were characterized in detail, and the assay application in a complex medium was validated. The proposed biosensing scheme as a proof-of-concept is expected to promote the application of oligonucleotide probes in basic research and medical diagnosis.     

Highly parallel single-molecule amplification approach based on agarose droplet polymerase chain reaction for efficient and cost-effective aptamer selection

We have developed a novel method for efficiently screening affinity ligands (aptamers) from a complex single-stranded DNA (ssDNA) library by employing single-molecule emulsion polymerase chain reaction (PCR) based on the agarose droplet microfluidic technology. In a typical systematic evolution of ligands by exponential enrichment (SELEX) process, the enriched library is sequenced first, and tens to hundreds of aptamer candidates are analyzed via a bioinformatic approach. Possible candidates are then chemically synthesized, and their binding affinities are measured individually. Such a process is time-consuming, labor-intensive, inefficient, and expensive. To address these problems, we have developed a highly efficient single-molecule approach for aptamer screening using our agarose droplet microfluidic technology. Statistically diluted ssDNA of the pre-enriched library evolved through conventional SELEX against cancer biomarker Shp2 protein was encapsulated into individual uniform agarose droplets for droplet PCR to generate clonal agarose beads. The binding capacity of amplified ssDNA from each clonal bead was then screened via high-throughput fluorescence cytometry. DNA clones with high binding capacity and low K(d) were chosen as the aptamer and can be directly used for downstream biomedical applications. We have identified an ssDNA aptamer that selectively recognizes Shp2 with a K(d) of 24.9 nM. Compared to a conventional sequencing-chemical synthesis-screening work flow, our approach avoids large-scale DNA sequencing and expensive, time-consuming DNA synthesis of large populations of DNA candidates. The agarose droplet microfluidic approach is thus highly efficient and cost-effective for molecular evolution approaches and will find wide application in molecular evolution technologies, including mRNA display, phage display, and so on.     

A homogeneous and noncompetitive immunoassay based on the enhanced fluorescence resonance energy transfer by leucine zipper interaction

Fluorescence resonance energy transfer (FRET) between two GFP variants is a powerful technique to describe protein-protein interaction in a biological system. However, it has a limitation that the two variants tethered to the respective proteins have to be in sufficient proximity upon binding, which is often difficult to attain by simple N- or C-terminal fusions. Here we describe a novel method to significantly enhance FRET between GFP variant-tagged proteins with the use of leucine zippers. For the homogeneous sandwich immunoassay of a high molecular weight antigen human serum albumin (HSA), two separate single-chain Fvs recognizing distant epitopes of HSA were respectively fused with fluorescence donor ECFP or acceptor EYFP, and FRET between the two was analyzed by fluorescence spectrometry. Because these two proteins did not give any detectable FRET uponantigen addition, we tethered each protein with a leucine zipper motif (c-Jun or FosB) at the C-terminus to help the neighborhood of the GFP variants. Upon antigen addition, the new pairs showed significant antigen-dependent FRET. By exchanging the binding domains, the method will find a range of applications for the assay of other proteins and their interactions in vitro or in vivo.     

结合运动仿真与阀信号检测的ESC HIL测试研究

为内置集成车辆姿态传感器的ESC能够进行完整的功能和故障安全策略测试,提出在硬件在环(HIL)仿真测试技术中融合运动仿真及电磁阀信号检测方法。三维运动转台通过模拟车辆三轴运动实现对车辆姿态传感器的运动激励;液压控制电磁阀信号检测单元通过霍尔传感器直接对线圈磁场强度进行感应,再通过参数标定实现对阀控制电流的准确采集,继而驱动液压调节器模型;基于悬架KC及轮胎动态试验等数据对整车系统模型进行参数化与调校,使之与实车特性具有较高的一致性;对功能测试内容及各类故障模式进行分析。构建的ESC HIL仿真测试系统平台能够为集成式ESC提供完整的虚拟整车运行环境,开发的测试评价规范可以作为对ESC控制进行评价的有效依据。     

Highly sensitive immunoassay based on immunogold-silver amplification and inductively coupled plasma mass spectrometric detection

In this work, we demonstrated a highly sensitive inductively coupled plasma mass spectrometric (ICPMS) method for the determination of human carcinoembryonic antigen (CEA), which combined the inherent high sensitivity of elemental mass spectrometric measurement with the signal amplification of catalytic silver deposition on immunogold tags. The silver amplification procedure was easy to handle and required cheap reagents, and the sensitivity was greatly enhanced to 60-fold after a 15 min silver amplification procedure. The experimental conditions, including detection of gold and silver by ICPMS, immunoassay parameters, silver amplification parameters, analytical performance, and clinical serum samples analysis, were investigated. The ICPMS Ag signal intensity depends linearly on the logarithm of the concentration of human CEA over the range of 0.07-1000 ng mL(-1) with a limit of detection (LOD, 3σ) of 0.03 ng mL(-1) (i.e., 0.15 pM). The LOD of the proposed method is around 2 orders of magnitude lower than that by the widely used enzyme-linked immunosorbent assay (ELISA) and 1 order of magnitude lower than that by clinical routine chemiluminescence immunoassay (CLIA) or time-resolved fluoroimmunoassay (TRFIA) and conventional ICPMS immunoassay. The present strategy was applied to the determination of human CEA in clinical human serum samples, and the results were in good agreement with those obtained by chemiluminescence immunoassay.     

基于NLMS自适应滤波的实时声音信号检测识别

在特定区域环境中,提出了一种基于NLMS自适应滤波的实时声音信号检测识别方法。该方法NLMS对滤波后的误差信号进行双门限检测,并以通过检测的疑似信号所持续的时间,作为其是否为目标信号的评判指标。通过对待处理声音文件的测试,该方法可以有效地去除非期望信号,保留目标信号,从而实现对目标信号的准确检测。     

时频分布的弱信号检测技术的研究及其应用

双线性时间-频率变换在时间域与频率域都具有较高的分辨率,有利于复杂背景条件下微弱瞬时信号的探测。研究发现,频谱图的分辨率较低,W igner-V ille分布存在很强的交叉项,不适合瞬时信号的检测。Cho i-W illiam s分布存在频率混叠及信息丢失现象,检测效果也不理想。提出了一种无混叠时频分布,能够避免CW D中的信息丢失,有效抑制交叉项,而且具有较高的分辨率。通过数字仿真与齿轮箱故障检测实例,证明新的时频分布能够有效检测复杂信号中的瞬时分量。     

Study of the monolithic integration of sub-bandgap detection, signal amplification and optical attenuation on a silicon photonic chip

We report the development of a silicon integrated circuit that combines conventional electronic circuitry with all-silicon optical waveguides, detectors and modulators. The circuit functions as an optical channel power leveller by amplifying current from a photodetector and feeding that current back to a modulator on the same waveguide. This article describes the use of local oxidation of silicon (LOCOS) for optical waveguide fabrication, the use of deep diffused wells to ensure electrical isolation between the forward biased modulator and detector diodes, and defect-engineering of the photodiodes to give them sufficient responsivity at the operating wavelength of 1.55 μm.     

Robust and highly sensitive fluorescence approach for point-of-care virus detection based on immunomagnetic separation

In this work, robust approach for a highly sensitive point-of-care virus detection was established based on immunomagnetic nanobeads and fluorescent quantum dots (QDs). Taking advantage of immunomagnetic nanobeads functionalized with the monoclonal antibody (mAb) to the surface protein hemagglutinin (HA) of avian influenza virus (AIV) H9N2 subtype, H9N2 viruses were efficiently captured through antibody affinity binding, without pretreatment of samples. The capture kinetics could be fitted well with a first-order bimolecular reaction with a high capturing rate constant k(f) of 4.25 × 10(9) (mol/L)(-1) s(-1), which suggested that the viruses could be quickly captured by the well-dispersed and comparable-size immunomagnetic nanobeads. In order to improve the sensitivity, high-luminance QDs conjugated with streptavidin (QDs-SA) were introduced to this assay through the high affinity biotin-streptavidin system by using the biotinylated mAb in an immuno sandwich mode. We ensured the selective binding of QDs-SA to the available biotin-sites on biotinylated mAb and optimized the conditions to reduce the nonspecific adsorption of QDs-SA to get a limit of detection low up to 60 copies of viruses in 200 μL. This approach is robust for application at the point-of-care due to its very good specificity, precision, and reproducibility with an intra-assay variability of 1.35% and an interassay variability of 3.0%, as well as its high selectivity also demonstrated by analysis of synthetic biological samples with mashed tissues and feces. Moreover, this method has been validated through a double-blind trial with 30 throat swab samples with a coincidence of 96.7% with the expected results.     

Chemiluminescence immunoassay based on dual signal amplification strategy of Au/mesoporous silica and multienzyme functionalized mesoporous silica

A chemiluminescent dual signal amplification strategy for the determination of α-fetoprotein (AFP) was proposed based on a sandwich immunoassay format. Monoclonal antibody of AFP immobilized on the gold nanoparticles doped mesoporous SiO₂ (Au/SiO₂) were prepared and used as a primary antibody. Horseradish peroxidase (HRP) and HRP-labeled secondary antibody (Ab₂) co-immobilized into the mesoporous SiO₂ nanoparticles (HRP-Ab₂/SiO₂) were used as the labeled immunological probe. Due to the high ratio surface areas and pore volumes of the mesoporous SiO₂, not only the amount of AFP monoclonal antibody but also the amount of the modified HRP and Ab₂ in HRP-Ab₂/SiO₂ were largely increased. Thus the chemiluminescent signal was amplified by using the system of luminol and H₂O₂ under the catalysis of HRP. Under the optimal conditions, two linear ranges for AFP were obtained from 0.01 to 0.5 ng mL⁻¹ and 0.5 to 100 ng mL⁻¹ with a detection limit of 0.005 ng mL⁻¹ (3σ). The fabricated signal amplification strategy showed an excellent promise for sensitive detection of AFP and other tumor markers.     

基于神经网络和功率谱的水中目标信号检测方法

将功率谱和神经网络相结合，应用于高海况、低信噪比条件下，水中目标信号的特征提取中．文中首先对信号进行功率谱估计，利用目标信号功率主要集中在低频部分的特点，提取低频信号的能量作为特征，然后利用人工神经网络对目标信号进行检测．利用不同浪级情况下海洋水压场的仿真信号数据，对某型目标舰船的水压信号进行了检测计算，验证了该方法的有效性，尤其是达到了在高海况、低信噪比条件下，对目标信号检测率比较高、虚警率比较低的效果．     

Sensitive and spatially multiplexed detection system based on dielectrophoretic manipulation of DNA-encoded particles used as immunoreactions platform

In this work, we designed a new immunodevice that combines competitive immunoreactions on the microparticles, accumulation of these particles by negative dielectrophoresis (n-DEP), and their subsequent capture through hybridization among single-stranded DNAs (ssDNAs). Two widely used pesticides, atrazine and bromopropylate, were used as target molecules to test the resulting simultaneous detection system. For sensing, we prepared two different sets of microparticles: one modified with atrazine-conjugated bovine serum albumin (BSA-2d) and ssDNA-J1(up) and the other with bromopropylate-conjugated aminodextran (AD-155) and ssDNA-J2(up). The microparticles were incubated in a mixture of analyte-specific antibody and analyte at different concentrations to trap the unreacted antibodies prior to being labeled with antibodies conjugated with a fluorescence molecule. A suspension containing both types of microparticles was introduced into an n-DEP device consisting of an interdigitated microarray (IDA) electrode and channel modified with ssDNA-J1(down) and ssDNA-J2(down), which are complementary to ssDNA-J1(up) and ssDNA-J2(up), respectively. The n-DEP force generated by applying AC voltage to the IDA electrode displaced the microparticles toward the encoded areas, causing them to rapidly accumulate on the upper surfaces. Hybridization allowed us to distinguish the microparticles and sense multiple analytes by spatial recognition in the DNA-encoded areas. The fluorescence intensity of the captured particles, which depends on analyte concentrations, was measured selectively by focusing on specific areas. The strategy is advantageous for sensitivity due to the equivalent trapping efficiency by DNA hybridization and large surface area of the microparticle for immunoreactions. The rapidity and simplicity were still supported by particle manipulation. Using this concept, we detect atrazine and bromopropylate simultaneously with limits of detection (LODs) of 0.2 μg·L(-1), which covered the maximum residue level (MRL) in food samples established the European Union (EU) and Japan Ministry of Health, Labor and Welfare (MHLW).     

Sensitive and selective detection of mercury (II) based on the aggregation of gold nanoparticles stabilized by riboflavin

Gold nanoparticles (AuNPs) can be stabilized by riboflavin against tris buffer-induced aggregation. However, in the presence of mercury (II) (Hg2+), riboflavin can be released from the AuNPs surface and the riboflavin-Hg2+ complex formed, leading to the aggregation of AuNPs in tris buffer. The aggregation extent depends on the concentration of Hg2+. Based on the aggregation extent, a simple and sensitive method of determining Hg2+ is developed. The method enables the detection of Hg2+ over the concentration range of 0.02-0.8 microM, with a detection limit (3sigma) of 14 nM, and shows excellent selectivity for Hg2+ over other metal ions (Cu2+, Co2+, Cd2+, Pb2+, Mg2+, Zn2+, Ag+, Ce3+, Ca2+, Al3+, K+). More importantly, the method avoids complicated surface modifications and tedious separation processes.     

Highly sensitive multiple microRNA detection based on fluorescence quenching of graphene oxide and isothermal strand-displacement polymerase reaction

A simple, highly sensitive, and selective multiple microRNA (miRNA) detection method based on the graphene oxide (GO) fluorescence quenching and isothermal strand-displacement polymerase reaction (ISDPR) was proposed. The capability to discriminate ssDNA and double-stranded nucleic acid structure coupled with the extraordinary fluorescence quenching of GO on multiple organic dye allows the proposed strategy to simultaneously and selectively detect several miRNA labeled with different dyes in the same solution, while the ISDPR amplification endows the detection method with high sensitivity. The strong interaction between ssDNA and GO led to the fluorescent ssDNA probe exhibiting minimal background fluorescence. Upon the recognition of specific target miRNA, an ISDPR was triggered to produce numerous massive specific DNA-miRNA duplex helixes, and a strong emission was observed due to the weak interaction between the DNA-miRNA duplex helix and GO. A miRNA biosensor down to 2.1 fM with a linear range of 4 orders of magnitude was obtained. Furthermore, the large planar surface of GO allows simultaneous quenching of several DNA probes with different dyes and produces a multiple biosensing platform with high sensitivity and selectivity, which has promising application in profiling the pattern of miRNA expression and biomedical research.     

基于飞秒激光与纳秒激光协同多光子诱导荧光的火焰中氢原子瞬态检测

为了准确测量碳氢燃料燃烧过程中原位氢原子,提出了飞秒-纳秒激光协同激发的氢原子瞬态在线探测策略。以243 nm的飞秒激光经过双光子过程将基态氢原子激发到2S能级,随后这些氢原子再被656 nm的纳秒激光激发到3P能级,通过探测3P-2S的荧光发射,实现了宽当量比范围的氢原子无干扰瞬态在线测量。实验结果表明：通过飞秒-纳秒协同的方式,可以有效降低激光光解产生的氢原子对原位氢原子探测的干扰。