Sensitive and homogeneous protein detection based on target-triggered aptamer hairpin switch and nicking enzyme assisted fluorescence signal amplification

Specific and sensitive detection of proteins in biotechnological applications and medical diagnostics is one of the most important goals for the scientific community. In this study, a new protein assay is developed on the basis of hairpin probe and nicking enzyme assisted signal amplification strategy. The metastable state hairpin probe with short loop and long stem is designed to contain a protein aptamer for target recognition. A short Black Hole Quencher (BHQ)-quenching fluorescence DNA probe (BQF probe) carrying the recognition sequence and cleavage site for the nicking enzyme is employed for fluorescence detection. Introduction of target protein into the assay leads to the formation change of hairpin probe from hairpin shape to open form, thus faciliating the hybridization between the hairpin probe and BQF probe. The fluorescence signal is amplified through continuous enzyme cleavage. Thrombin is used as model analyte in the current proof-of-concept experiments. This method can detect thrombin specifically with a detection limit as low as 100 pM. Additionally, the proposed protein detection strategy can achieve separation-free measurement, thus eliminating the washing steps. Moreover, it is potentially universal because hairpin probe can be easily designed for other proteins by changing the corresponding aptamer sequence.     

Spatial fluorescence cross-correlation spectroscopy

We present an alternative method for diffusion measurements of fluorescent species in solution by use of confocal microscopy and fluorescence correlation spectroscopy techniques. It consists of making a time and spatial dual correlation in which one detects the fluorescence signals from two nearby separate confocal volumes and cross correlates them. To improve the spatial discrimination between the two confocal volumes we propose filtering of fluorescence photocounts by rejecting the fluorescence background, which corresponds to particles located far from the center of the detection volumes.     

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.     

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.     

Two-beam fluorescence cross-correlation spectroscopy in an electrophoretic mobility shift assay

Two-beam fluorescence cross-correlation spectroscopy (FCCS) was used to resolve the bound and unbound fractions of fluorescently labeled single-stranded DNA (ssDNA) in a ssDNA-protein complex as the analyte solution flowed continuously through an electrophoresis capillary. Cross-correlation of the single molecule fluorescence from two spatially separate excitation laser beams resulted in cross-correlation functions that consisted of well-resolved peaks characteristic of the different electrophoretic flow velocities of the bound and unbound ssDNA. This decoupled the molecular parameters of the bound and unbound ssDNA used to model the cross-correlation function, which enabled the relative concentrations to be determined without prior knowledge of the pure-component cross-correlation functions, as would be required in an analogous autocorrelation analysis. The relative concentrations of the bound and unbound ssDNA were determined by two-beam FCCS within 2-6% precision, even for samples that contained as little as 5% unbound ssDNA, and were consistent with the results obtained by capillary electrophoresis (CE) separation of the same samples. Data sufficient to obtain these results was acquired in 10-15 s per sample. Fluorescently labeled poly(dT)39 complexed with the single stranded DNA binding protein of Escherichia coli served as the model system. The measured dissociation constant of 2.5+/-0.9 nM agreed with the literature value for this complex within experimental error. The CE/two-beam FCCS experiment described here is part of a family of techniques that use single molecule fluorescence detection to resolve different components in an electrophoresis system. Advantages of these methods relative to separations-based CE include enhanced sensitivity, the potential for higher speed analyses, elimination of the sample plug injection step, and the ability to carry out the analysis in shorter flow channels.     

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.     

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.     

Multiple-fiber probe design for fluorescence spectroscopy in tissue

The fiber-optic probe is an essential component of many quantitative fluorescence spectroscopy systems, enabling delivery of excitation light and collection of remitted fluorescence in a wide variety of clinical and laboratory situations. However, there is little information available on the role of illumination--collection geometry to guide the design of these components. Therefore we used a Monte Carlo model to investigate the effect of multifiber probe design parameters--numerical aperture, fiber diameter, source--collection fiber separation distance, and fiber-tissue spacer thickness--on light propagation and the origin of detected fluorescence. An excitation wavelength of 400 nm and an emission wavelength of 630 nm were simulated. Noteworthy effects included an increase in axial selectivity with decreasing fiber size and a transition with increasing fiber-tissue spacer size from a subsurface peak in fluorophore sensitivity to a nearly monotonic decrease typical of single-fiber probes. We provide theoretical evidence that probe design strongly affects tissue interrogation. Therefore application-specific customization of probe design may lead to improvements in the efficacy of fluorescence-based diagnostic devices.     

Calibration of probe volume in fluorescence correlation spectroscopy

In fluorescence correlation spectroscopy (FCS), an accurate evaluation of the probe volume is the basis of correct interpretation of experimental data and solution of an appropriate diffusion model. Poor fitting convergence has been a problem in the determination of the dimensional parameters, the beam radius, omega, and the distance along the optical axis of the probe volume, l. In this work, the instability of fitting during the calibration process is investigated by examining the chi(2) surfaces. We demonstrate that the minimum of chi(2) in the omega dimension is well defined for both converging and diverging data. The difficulty of fitting comes from the l dimension. The uncertainty in l could be significantly larger than that in omega, as determined by F-statistics. A modified calibration process is recommended based on examining two data treatment methods, combining several short data sets into a single long run and averaging the correlation functions of several short data sets. It is found that by using the mean of several converging correlation functions from short data sets instead of a long time correlation, more stable and consistent dimensional parameters are extracted to define the probe volume.     

Spatial two-photon fluorescence cross-correlation spectroscopy for controlling molecular transport in microfluidic structures

The increasing availability of microfluidic systems of various geometries and materials for the downscaling of chemical or biochemical processes raises a strong demand for adequate techniques to precisely determine flow parameters and to control fluid and particle manipulation. Of all readout parameters, fluorescence analysis of the fluid or suspended particles is particularly attractive, as it can be employed without mechanical interference and with a sensitivity high enough to detect single molecules in aqueous environments. In this study, we present the determination of flow parameters, such as velocity and direction, in microstructured channels by fluorescence correlation spectroscopy (FCS), a method based on single molecule spectroscopy carried out in confocal optical setups. Different modes of FCS, such as auto- and dual-beam cross-correlation techniques by one- and two-photon excitation, are discussed. Known advantages of two-photon excitation, such as highly restricted detection volumes and low scattering background, are shown to be particularly valuable for measurements in tiny channel systems. Although conventional autocorrelation is sufficient for describing the velocity of single molecules, dual-beam cross-correlation allows the separation of isotropic and anisotropic dynamics, for example, to monitor flow directions or to discriminate against photophysical effects that could be mistaken for mobility parameters. It can be shown that time-gated two-photon excitation in the dual-beam mode significantly lowers the undesired cross-talk between the two measurement volumes. Finally, some applications, such as the calibration of microfluidic sorting units and flow profiling, are demonstrated.     

Two-beam fluorescence cross-correlation spectroscopy for simultaneous analysis of positive and negative ions in continuous-flow capillary electrophoresis

Two-beam fluorescence cross-correlation spectroscopy was used for multicomponent electrophoretic analysis of positive and negative ions flowing continuously, and in opposite directions, through a polymer-coated electrophoresis capillary. Cross-correlation analysis of the fluorescence monitored from two spatially offset microscopic detection volumes revealed the magnitude and direction of the electrophoretic flow velocity of each analyte. This enabled resolution of a three-component mixture containing nanomolar concentrations of cationic rhodamine 6G and anionic 5-carboxytetramethylrhodamine (TAMRA) and TAMRA-labeled single-stranded DNA in aqueous buffer. The relative concentrations of each analyte, determined from the cross-correlation analysis, agreed with known values within experimental error. This technique is sensitive to molecular processes occurring on a millisecond time scale and will prove useful for monitoring rapid fluctuations in the magnitude and direction of the electrophoretic flow velocity caused by spontaneous chemical reactions or conformational transformations at thermodynamic equilibrium.     

Single-stranded DNA binding protein-assisted fluorescence polarization aptamer assay for detection of small molecules

Here, we describe a new fluorescence polarization aptamer assay (FPAA) strategy which is based on the use of the single-stranded DNA binding (SSB) protein from Escherichia coli as a strong FP signal enhancer tool. This approach relied on the unique ability of the SSB protein to bind the nucleic acid aptamer in its free state but not in its target-bound folded one. Such a feature was exploited by using the antiadenosine (Ade)-DNA aptamer (Apt-A) as a model functional nucleic acid. Two fluorophores (fluorescein and Texas Red) were introduced into different sites of Apt-A to design a dozen fluorescent tracers. In the absence of the Ade target, the binding of the labeled aptamers to SSB governed a very high fluorescence anisotropy increase (in the 0.130-0.200 range) as the consequence of (i) the large global diffusion difference between the free and SSB-bound tracers and (ii) the restricted movement of the dye in the SSB-bound state. When the analyte was introduced into the reaction system, the formation of the folded tertiary structure of the Ade-Apt-A complex triggered the release of the labeled nucleic acids from the protein, leading to a strong decrease in the fluorescence anisotropy. The key factors involved in the fluorescence anisotropy change were considered through the development of a competitive displacement model, and the optimal tracer candidate was selected for the Ade assay under buffer and realistic (diluted human serum) conditions. The SSB-assisted principle was found to operate also with another aptamer system, i.e., the antiargininamide DNA aptamer, and a different biosensing configuration, i.e., the sandwich-like design, suggesting the broad usefulness of the present approach. This sensing platform allowed generation of a fluorescence anisotropy signal for aptamer probes which did not operate under the direct format and greatly improved the assay response relative to that of the most previously reported small target FPAA.     

一种基于广义互相关的水声直扩信号检测方法

直扩信号因其低功率谱密度特性使得检测十分困难,针对传统自相关法在低信噪比条件下检测性能急剧下降的问题,在分析了直扩信号自相关特性的前提下,提出一种基于广义互相关估计的直扩信号检测方法.首先对接收信号分段并对相邻信号段分别进行广义互相关估计,估计结果采用二阶矩非相干积累,提取相关峰获得检测统计量,将检测统计量与门限比较,...     

Fluorescent detection of ATP based on signaling DNA aptamer attached silica nanoparticles

Novel methods for rapid, sensitive and low-cost biomolecule detection have attracted particular interest because of their wide use in medical diagnostics, food inspection and biomedical research applications. In this work, we report a simple and efficient silica nanoparticle (NP)-based fluorescent assay for ATP detection. It takes advantage of the washing and separation properties of NPs and the structure-switch property of DNA aptamers, resulting in fluorescence change of the supernatant in the presence of targets. A linear response for ATP detection was observed from 0 to 6?mM with a detection limit of ～34?μM. This detection strategy could be generalized to other aptamer-based detection systems.     

Spectrally resolved fluorescence correlation spectroscopy based on global analysis

Multicolor fluorescence correlation spectroscopy has been recently developed to study chemical interactions of multiple chemical species labeled with spectrally distinct fluorophores. In the presence of spectral overlap, there exists a lower detectability limit for reaction products with multicolor fluorophores. In addition, the ability to separate bound product from reactants allows thermodynamic properties such as dissociation constants to be measured for chemical reactions. In this report, we utilize a spectrally resolved two-photon microscope with single-photon counting sensitivity to acquire spectral and temporal information from multiple chemical species. Further, we have developed a global fitting analysis algorithm that simultaneously analyzes all distinct auto- and cross-correlation functions from 15 independent spectral channels. We have demonstrated that the global analysis approach allows the concentration and diffusion coefficients of fluorescent particles to be resolved despite the presence of overlapping emission spectra.     

Fiber-optic laser-induced fluorescence probe for the detection of environmental pollutants

Laser-induced fluorescence (LIF) spectroscopy in combination with fiber optics is shown to be a powerful tool for qualitative and quantitative diagnostics of environmental pollutants in water and soil. Timeintegrated data accumulation of the LIF signals in early and late time windows with respect to the excitation pulse simplifies the method so that it becomes attractive for practical applications. Results from field measurements are reported, as oil contaminations under a gas station and in an industrial sewer system are investigated. A KrF-excimer laser and a hydrogen Raman shifter can be applied for multiwavelength excitation. This allows a discrimination between benzene, toluene, xylene, and ethylbenzene aromatics and polycyclic aromatic hydrocarbon molecules in the samples under investigation. For a rough theoretical approach, a computer simulation is developed to describe the experimental results.     

Element-specific detection in capillary electrophoresis using X-ray fluorescence spectroscopy

X-ray fluorescence spectroscopy is demonstrated here as a novel, element-specific detector for capillary electrophoresis. Monochromatic 10 keV X-rays from a synchrotron light source are used to excite core electrons, causing emission of characteristic Kalpha X-ray fluorescence (XRF) lines. Using this technique, XRF energies provide elemental identification, while XRF intensities can be used to quantitate the metal composition of each eluent. An X-ray transparent polymer coupling is used to create a window for the on-line, X-ray detection. This coupling contributes no measurable extra-column variance, and electrophoretic mobilities for the metal complexes used as model solutes are highly reproducible. The combination of XRF detection with capillary electrophoresis (CE-XRF) creates the first on-line detection system that is element-specific, nondestructive, and directly applicable to a broad range of applications including nonelectroactive species. CE-XRF is successfully demonstrated here for high binding-constant complexes of Fe(III), Co(II), Cu(II), and Zn(II). Within a single injection, electropherograms are obtained for each element of interest, with the element identity obtained directly from the emission energy. In contrast with ICPMS, this detection technique is directly on-line and does not require volatilization of the eluent. As a result, element-specific detection is not limited by the sample or the buffer volatility or atomization efficiency. Simultaneous XRF and UV absorbance detection can be used to provide an on-line determination of metal/chelate ratios. Although XRF detection limits are presently only in the 0.1 mM (0.5 ng) range, both collection geometry and incident intensity have yet to be optimized. Further optimization is expected to enhance this detection limit by another 2-3 orders of magnitude. As a result, the advent of XRF detection combined with the separating power of CE presents new possibilities for on-line, element-specific analysis.     

Raman spectroscopy and microscopy based on mechanical force detection

The Raman effect is typically observed by irradiating a sample with an intense light source and detecting the minute amount of frequency shifted scattered light. We demonstrate that Raman molecular vibrational resonances can be detected directly through an entirely different mechanism-namely, a force measurement. We create a force interaction through optical parametric down conversion between stimulated, Raman excited, molecules on a surface and a cantilevered nanometer scale probe tip brought very close to it. Spectroscopy and microscopy on clusters of molecules have been performed. Single molecules within such clusters are clearly resolved in the Raman micrographs. The technique can be readily extended to perform pump probe experiments for measuring inter- and intramolecular couplings and conformational changes at the single molecule level.     

Determination of nitrite in waters by microplate fluorescence spectroscopy and HPLC with fluorescence detection.

A selective and versatile fluorescence spectroscopic method for the determination of nitrite in waters has been developed. Nitrite reacts in the presence of mineral acids with the nonfluorescent N-methyl-4-hydrazino-7-nitrobenzofurazan forming N-methyl-4-amino-7-nitrobenzofurazan, which can be detected by fluorescence spectroscopy with an excitation maximum at lambda = 468 nm and an emission maximum at lambda = 537 nm in acetonitrile. Three new methods based on this reaction have been developed: Direct fluorescence spectroscopy, HPLC/fluorescence, or HPLC with UV/vis detector may be selected as detection techniques. On microplates, high-throughput fluorescence spectroscopy is achieved, while HPLC/fluorescence provides lower limits of detection, and HPLC with UV/vis detection enables evaluation of the reaction with standard instrumentation. Different water samples were investigated using all detection modes, and a photometric standard procedure was successfully employed to validate the new methods with an independent technique.     

Attomole-level protein fingerprinting based on intrinsic peptide fluorescence

Protein identification has relied heavily on proteolytic analysis, but current techniques are often slow and generally consume large quantities of valuable protein sample. We report the development of a rapid, ultralow volume protein analysis strategy based on tryptic digestion within the tip of a 1.5-microm capillary channel followed by separation of the proteolytic fragments using capillary electrophoresis (CE). Two-photon excitation is used to probe the intrinsic fluorescence of peptide fragments through "deep-UV" excitation of aromatic amino acid residues at the outlet of the CE channel. Detection limits using this technique are 0.7, 2.4, and 23 amol for the aromatic amino acids tryptophan, tyrosine, and phenylalanine, respectively. In these studies, we demonstrate the capacity to differentiate bovine and yeast cytochrome c variants using less than 15 amol of protein through tryptic fingerprinting. Moreover, the detection of a single amino acid substitution between bovine and canine cytochrome c illustrates the sensitivity of this approach to minor differences in protein sequence. The 2-pL sample volume required for this on-column tryptic digestion is, to our knowledge, the smallest yet reported for a proteolytic assay.