A simple quenching method for fluorescence background reduction and its application to the direct, quantitative detection of specific mRNA

New genome sequence information is rapidly increasing the number of nucleic acid (NA) targets of use for characterizing and treating diseases. Detection of these targets by fluorescence-based assays is often limited by fluorescence background from unincorporated or unbound probes that are present in large excess over the target. To solve this problem, energy transfer-based probes have been developed and used to reduce the fluorescence from unbound probes. Although these probes have revolutionized NA target detection, their use requires scrupulous attention to design constraints, extensive probe quality control, and individually optimized experimental conditions. Here, we describe a simpler background reduction approach using singly labeled quencher oligomers to suppress excess unbound probe fluorescence following probe-target hybridization. A second limitation of most fluorescence-based NA target detection and quantification assays is the requirement for enzymatic amplification of target or signal for sensitivity. Amplification steps make quantification of original target copy number problematic because of variations in amplification efficiencies between the sequence targets and the experimental conditions. To avoid amplification, we coupled our quenching approach to a two-color NA assay with correlated, two-color, single-molecule fluorescence detection. We demonstrate a >100-fold background reduction and detection of targets present at concentrations as low as 100 fM using the two-color assay. The application of this technique to the detection and quantification of specific mRNA sequences enabled us to estimate beta-actin copy numbers in cell-derived total RNA without an amplification step.     

杂交链式反应技术结合纳米材料用于核酸检测的研究进展

核酸检测在疾病早期诊断、分期、治疗监测、预后判断以及产前基因诊断等许多方面有着重要意义,然而目前对核酸的检测技术不仅存在耗时长、成本高等问题,而且工作量大、操作繁琐,严重影响其在诊断中的应用。杂交链式反应(hybridization chain reaction,HCR)是一种新型信号扩增技术,它通过单链DNA(ssDNA)引发两种稳定的DNA发夹探针在恒温下发生杂交反应,从而放大检测信号,实现选择性检测靶分子的目的。不仅如此,HCR还可以结合不同的纳米材料用于检测各种目标物如核酸、蛋白质、细胞、金属离子等等。对HCR技术结合几种常见的纳米材料(银纳米颗粒、金纳米颗粒、氧化石墨烯以及几种其他纳米材料)用于核酸的检测进行了总结,并对今后的发展趋势进行了展望。     

Nanofluidic preconcentration and detection of nanoparticles

The fast detection and characterization of nanoparticles, such as viruses or environmental pollutants, are important in fields ranging from biosensing to quality control. However, most existing techniques have practical throughput limitations, which significantly limit their applicability to low analyte concentrations. Here, we present an integrated nanofluidic scheme for preconcentration and subsequent detection of nanoparticle samples within a continuous flow-through system. Using a Brownian ratchet mechanism, we increase the nanoparticle concentration ～27-fold. Single nanoparticles are subsequently detected and characterized by optical heterodyne interferometry. A wide range of potential applications can be foreseen, including real-time analysis of clinically relevant virus samples and contamination control of processing fluids used in the semiconductor industry.     

Gold nanoparticles as selective and concentrating probes for samples in MALDI MS analysis

MALDI mass spectrometry is used widely in various fields because it has the characteristics of speed, ease of use, high sensitivity, and wide detectable mass range, but suppression effects between analyte molecules and interference from the sample matrix frequently arise during MALDI analysis. The suppression effects can be avoided if target species are isolated from complicated matrix solutions in advance. Herein, we proposed a novel method for achieving such a goal. We describe a strategy that uses gold nanoparticles to capture charged species from a sample solution. Generally, ionic agents, such as anionic or cationic stabilizers, encapsulate gold nanoparticles to prevent their aggregation in solution. These charged stabilizers at the surface of the gold particles are capable of attracting oppositely charged species from a sample solution through electrostatic interactions. We have employed this concept to develop nanoparticle-based probes that selectively trap and concentrate target species in sample solutions. Additionally, to readily isolate them from solution after attracting their target species, we used gold nanoparticles that are adhered to the surface of magnetic particles through S-Au bonding. A magnet can then be employed to isolate the Au@magnetic particles from the solution. The species trapped by the isolated particles were then characterized by MALDI MS after a simple washing. We demonstrate that Au@magnetic particles having negatively charged surfaces are suitable probes for selectively trapping positively charged proteins from aqueous solutions. In addition, we have employed Au@magnetic particle-based probes successfully to concentrate low amounts of peptide residues from the tryptic digest products of cytochrome c (10(-7) M).     

Biosensing with plasmonic nanosensors

Recent developments have greatly improved the sensitivity of optical sensors based on metal nanoparticle arrays and single nanoparticles. We introduce the localized surface plasmon resonance (LSPR) sensor and describe how its exquisite sensitivity to size, shape and environment can be harnessed to detect molecular binding events and changes in molecular conformation. We then describe recent progress in three areas representing the most significant challenges: pushing sensitivity towards the single-molecule detection limit, combining LSPR with complementary molecular identification techniques such as surface-enhanced Raman spectroscopy, and practical development of sensors and instrumentation for routine use and high-throughput detection. This review highlights several exceptionally promising research directions and discusses how diverse applications of plasmonic nanoparticles can be integrated in the near future.     

Single-molecule kinetics of nanoparticle catalysis

Owing to their structural dispersion, the catalytic properties of nanoparticles are challenging to characterize in ensemble-averaged measurements. The single-molecule approach enables studying the catalysis of nanoparticles at the single-particle level with real-time single-turnover resolution. This article reviews our single-molecule fl uorescence studies of single Au-nanoparticle catalysis, focusing on the theoretical formulations for extracting quantitative reaction kinetics from the single-turnover resolution catalysis trajectories. We discuss the single-molecule kinetic formulism of the Langmuir-Hinshelwood mechanism for heterogeneous catalysis, as well as of the two-pathway model for product dissociation reactions. This formulism enables the quantitative evaluation of the heterogeneous reactivity and the differential selectivity of individual nanoparticles that are usually hidden in ensemble measurements. Extension of this formulism to single-molecule catalytic kinetics of oligomeric enzymes is also discussed.      

Optical detection of single non-absorbing molecules using the surface plasmon resonance of a gold nanorod

Existing methods for the optical detection of single molecules require the molecules to absorb light to produce fluorescence or direct absorption signals. This limits the range of species that can be detected, because most molecules are purely refractive. Metal nanoparticles or dielectric resonators can be used to detect non-absorbing molecules because local changes in the refractive index produce a resonance shift. However, current approaches only detect single molecules when the resonance shift is amplified by a highly polarizable label or by a localized precipitation reaction on the surface of a nanoparticle. Without such amplification, single-molecule events can only be identified in a statistical way. Here, we report the plasmonic detection of single molecules in real time without the need for labelling or amplification. Our sensor consists of a single gold nanorod coated with biotin receptors, and the binding of single proteins is detected by monitoring the plasmon resonance of the nanorod with a sensitive photothermal assay. The sensitivity of our device is ～700 times higher than state-of-the-art plasmon sensors and is intrinsically limited by spectral diffusion of the surface plasmon resonance.     

High-throughput detection and sizing of individual low-index nanoparticles and viruses for pathogen identification

Rapid, chip-scale, and cost-effective single particle detection of biological agents is of great importance to human health and national security. We report real-time, high-throughput detection and sizing of individual, low-index polystyrene nanoparticles and H1N1 virus. Our widefield, common path interferometer detects nanoparticles and viruses over a very large sensing area, orders of magnitude larger than competing techniques. We demonstrate nanoparticle detection and sizing down to 70 nm in diameter. We clearly size discriminate nanoparticles with diameters of 70, 100, 150, and 200 nm. We also demonstrate detection and size characterization of hundreds of individual H1N1 viruses in a single experiment.     

Improving the signal sensitivity and photostability of DNA hybridizations on microarrays by using dye-doped core-shell silica nanoparticles

The development of new highly sensitive and selective methods for microarray-based analysis is a great challenge because, for many bioassays, the amount of genetic material available for analysis is extremely limited. Currently, imaging and detection of DNA microarrays are based primarily on the use of organic dyes. To overcome the problems of photobleaching and low signal intensities of organic dyes, we developed a new class of silica core-shell nanoparticles that encapsulated with cyanine dyes and applied the dye-doped nanoparticles as labeling in the DNA microarray-based bioanalysis. The developed nanoparticles have core-shell structure containing 15-nm Au colloidal cores with 95 dye-alkanethiol (dT)20 oligomers chemisorbed on the each Au particle surface and 10-15-nm silica coatings bearing thiol functional groups. To be utilized for microarray detection, the dye-doped nanoparticles were conjugated with DNA signaling probes by using heterobifunctional cross-linker. The prepared nanoparticle conjugates are stable in both aqueous electrolytes and organic solvents. Two-color DNA microarray-based detection was demonstrated in this work by using Cy3- and Cy5-doped nanoparticles in sandwich hybridization. The use of the fluorophore-doped nanoparticles in high-throughput microarray detection reveals higher sensitivity with a detection limit of 1 pM for target DNA in sandwich hybridization and greater photostable signals than the direct use of organic fluorophore as labeling. A wide dynamic range of approximately 4 orders of magnitude was also found when the dye-doped nanoparticles were applied in microarray-based DNA bioanalysis. In addition, the use of these dye-doped nanoparticles as the labeling in hybridization also improved the differentiation of single-nucleotide polymorphisms. This work offers promising prospects for applying dye-doped nanoparticles as labeling for gene profiling based on DNA microarray technology.     

Optimized threshold selection for single-molecule two-color fluorescence coincidence spectroscopy

Two-color coincidence detection is a single-molecule fluorescence technique that is capable of resolving subpopulations of biomolecular complexes at very low concentrations. In this paper, we have developed a method that automatically determines appropriate thresholds for the analysis of sets of two-color coincidence data. This has the distinct advantage of allowing the rapid determination of optimized thresholds in a reproducible fashion. The trade-offs involved in such selections are that the thresholds should be high enough both to exceed the background photon count rates and to ensure a low rate of chance coincident events and that they should be low enough to give reasonably high rates of fluorescence events. Previously, thresholds were selected by judgment to balance these various separate considerations. The method reported in this paper incorporates the three factors into the maximization of a single value derived from the data as a function of the thresholds used in the two channels. The value that is maximized is a ratio of event rates, specifically the rate of coincident events above that expected by chance, divided by the total event rate; this is called the association quotient. In this paper, we demonstrate that maximization of the association quotient selects appropriate thresholds for data derived from dual-labeled duplex DNA samples over a range of concentrations and laser powers. This method should allow the application of two-color coincidence detection to more complex biological systems and cells where the sample concentration and background levels are more variable and where it is not possible to run separate control experiments to determine the latter statistics.     

Aptamer-conjugated nanoparticles for selective collection and detection of cancer cells

We have developed a method for the rapid collection and detection of leukemia cells using a novel two-nanoparticle assay with aptamers as the molecular recognition element. An aptamer sequence was selected using a cell-based SELEX strategy in our laboratory for CCRF-CEM acute leukemia cells that, when applied in this method, allows for specific recognition of the cells from complex mixtures including whole blood samples. Aptamer-modified magnetic nanoparticles were used for target cell extraction, while aptamer-modified fluorescent nanoparticles were simultaneously added for sensitive cell detection. Combining two types of nanoparticles allows for rapid, selective, and sensitive detection not possible by using either particle alone. Fluorescent nanoparticles amplify the signal intensity corresponding to a single aptamer binding event, resulting in improved sensitivity over methods using individual dye-labeled probes. In addition, aptamer-modified magnetic nanoparticles allow for rapid extraction of target cells not possible with other separation methods. Fluorescent imaging and flow cytometry were used for cellular detection to demonstrate the potential application of this method for medical diagnostics.     

Nitrilotriacetic acid-coated magnetic nanoparticles as affinity probes for enrichment of histidine-tagged proteins and phosphorylated peptides

We herein demonstrate superparamagnetic Fe3O4 nanoparticles coated with nitrilotriacetic acid derivative (NTA) that can bind with different immobilized metal ions are capable of probing diverse target species. Immobilized Ni(II) on the surfaces of the NTA-magnetic nanoparticles have the capability of selectively trapping histidine (His)-tagged proteins such as a mutated streptopain tagged with 6x His, i.e., C192S (MW approximately 42 kDa), from cell lysates. Enrichment was achieved by vigorously mixing the sample solution and the nanoparticles by pipetting in and out of a sample vial for only 30 s. After enrichment, the probe-target species could be readily isolated by magnetic separation. We also characterized the proteins enriched on the affinity probes using on-probe tryptic digestion under microwave irradiation for only 2 min, followed by matrix-assisted laser desorption/ionization mass spectrometry analysis. Using this enrichment and tryptic digestion, the target species can be rapidly enriched and characterized, reducing the time required for carrying out the complete analysis to less than 10 min. Furthermore, when either Zr(IV) or Ga (III) ions are immobilized on the surfaces of the NTA-magnetic nanoparticles, the nanoparticles have the capability of selectively enriching phosphorylated peptides from tryptic digests of alpha-, beta-caseins, and diluted milk. The detection limit for the tryptic digests of alpha- and beta-caseins is approximately 50 fmol.     

Combination of functionalized nanoparticles and polymerase chain reaction-based method for SARS-CoV gene detection

Rapid and sensitive detection of SARS associated coronavirus is critical for early diagnosis and control of severe acute respiratory syndrome. This study describes a method for the detection of SARS associated coronavirus gene by the combination of functionalized nanoparticles and PCR-based assay. In this method, the target cDNA of SARS associated coronavirus was firstly captured and enriched from the mixture of target cDNA and non-target cDNA by the use of the functionalized silica coated superparamagnetic nanoparticles. Additionally, the enriched target cDNA were amplified through a general symmetry PCR and then was selectively isolated from the double strands PCR products by applying the silica coated superparamagnetic nanoparticles again. Finally, we detected the amplified target cDNA by employing the functionalized silica coated fluorescent nanoparticles as signaling probes with a sandwich hybridization format. The results show that the target cDNA can be assayed successfully with a detection limit of 2.0 x 10(3) copies and the nonspecific amplification could be inhibited. In addition, the detection procedure is rapid and can be completed in less than 6 h. Our results suggest that this approach will provide promising prospects for other pathogens detection.     

Probing single molecules in single living cells

Single-molecule detection in single living cells has been achieved by using confocal fluorescence microscopy and externally tagged probe molecules. The intracellular background fluorescence is substantially higher than that in aqueous buffer, but this background is continuous and stable and does not significantly interfere with the measurement of single-molecule photon bursts. As a result, single-molecule data have been obtained on three types of fluorescent probes at spatially resolved locations (e.g., cytoplasm and nucleus) inside human HeLa cells. First, the iron transport protein transferrin labeled with tetramethylrhodamine undergoes rapid receptor-mediated endocytosis, and single transferrin molecules are detected inside living cells. Second, the cationic dye rhodamine 6G (R6G) enters cultured cells by a potential-driven process, and single R6G molecules are observed as intense photon bursts when they move in and out of the intracellular laser beam. Third, we report results on synthetic oligonucleotides that are tagged with a fluorescent dye and are taken up by living cells via a passive, nonendocytic pathway.     

Homogeneous detection of nucleic acids based upon the light scattering properties of silver-coated nanoparticle probes

Herein we report the development of a simple, rapid, homogeneous, and sensitive detection system for DNA based on the scattering properties of silver-amplified gold nanoparticle probes. The assay uses DNA-functionalized magnetic particle probes that act as scavengers for target DNA, which can be collected via a magnetic field. Once the DNA targets are isolated from the initial sample, they are sandwiched via hybridization by a second set of probes. The latter probes are 13-nm gold nanoparticles modified with a different target complementary DNA. Excess probes are removed through repetitive washing steps. The gold particles are dispersed in solution by dehybridization, corresponding to an assumed 1:1 ratio with the target DNA. Electroless deposition of silver on the surface of the gold probes results in particle growth, which increases their scattering efficiency with time. The scattering efficiency and the extinction signatures of the particle sizes are monitored as a function of time and correlated with target concentration. The limit of detection for this novel assay was determined to be 10 fM.     

Single-molecule analysis of ultradilute solutions with guided streams of 1--microm water droplets

We describe instrumentation for real-time detection of single-molecule fluorescence in guided streams of 1-mum (nominal) water droplets. In this technique, target molecules were confined to droplets whose volumes were comparable with illumination volumes in diffraction-limited fluorescence microscopy and guided to the waist of a cw probe laser with an electrostatic potential. Concentration detection limits for Rhodamine 6G in water were determined to be ~1 fM, roughly 3 orders of magnitude lower than corresponding limits determined recently with diffraction-limited microscopy techniques for a chemical separation of similar dyes. In addition to its utility as a vehicle for probing single molecules, instrumentation for producing and focusing stable streams of 1-2-mum-diameter droplets may have other important analytical applications as well.     

Probes for detection of specific DNA sequences at the single-molecule level

A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe-target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules.     

A scheme for detecting every single target molecule with surface-enhanced Raman spectroscopy

Surface-enhanced Raman spectroscopy (SERS) is now a well-established technique for the detection, under appropriate conditions, of single molecules (SM) adsorbed on metallic nanostructures. However, because of the large variations of the SERS enhancement factor on the surface, only molecules located at the positions of highest enhancement, so-called hot-spots, can be detected at the single-molecule level. As a result, in all SM-SERS studies so far only a small fraction, typically less than 1%, of molecules are actually observed. This complicates the analysis of such experiments and means that trace detection via SERS can in principle still be vastly improved. Here we propose a simple scheme, based on selective adsorption of the target analyte at the SERS hot-spots only, that allows in principle detection of every single target molecule in solution. We moreover provide a general experimental methodology, based on the comparison between average and maximum (single molecule) SERS enhancement factors, to verify the efficiency of our approach. The concepts and tools introduced in this work can readily be applied to other SERS systems aiming for detection of every single target molecule.     

Correlation-matrix analysis of two-color coincidence events in single-molecule fluorescence experiments

We introduce a robust and relatively easy-to-use method to evaluate the quality of two-color (or more) fluorescence coincidence measurements based on close investigation of the coincidence correlation-matrix. This matrix contains temporal correlations between the number of detected bursts in individual channels and their coincidences. We show that the Euclidian norm of a vector Γ derived from elements of the correlation matrix takes a value between 0 and 2 depending on the relative coincidence frequency. We characterized the Γ-norm and its dependence on various experimental conditions by computer simulations and fluorescence microscopy experiments. Single-molecule experiments with two differently colored dye molecules diffusing freely in aqueous solution, a sample that generates purely random coincidence events, return a Γ-norm less than one, depending on the concentration of the fluorescent dyes. As perfect coincidence sample we monitored broad autofluorescence of 2.8 μm beads and determined the Γ-norm to be maximal and close to two. As in realistic diagnostic applications, we show that two-color coincidence detection of single-stranded DNA molecules, using differently labeled Molecular Beacons hybridizing to the same target, reveal a value between one and two representing a mixture of an optimal coincidence sample and a sample generating random coincidences. The Γ-norm introduced for data analysis provides a quantifiable measure for quickly judging the outcome of single-molecule coincidence experiments and estimating the quality of detected coincidences.     

Detecting single viruses and nanoparticles using whispering gallery microlasers

There is a strong demand for portable systems that can detect and characterize individual pathogens and other nanoscale objects without the use of labels, for applications in human health, homeland security, environmental monitoring and diagnostics. However, most nanoscale objects of interest have low polarizabilities due to their small size and low refractive index contrast with the surrounding medium. This leads to weak light-matter interactions, and thus makes the label-free detection of single nanoparticles very difficult. Micro- and nano-photonic devices have emerged as highly sensitive platforms for such applications, because the combination of high quality factor Q and small mode volume V leads to significantly enhanced light-matter interactions. For example, whispering gallery mode microresonators have been used to detect and characterize single influenza virions and polystyrene nanoparticles with a radius of 30 nm (ref. 12) by measuring in the transmission spectrum either the resonance shift or mode splitting induced by the nanoscale objects. Increasing Q leads to a narrower resonance linewidth, which makes it possible to resolve smaller changes in the transmission spectrum, and thus leads to improved performance. Here, we report a whispering gallery mode microlaser-based real-time and label-free detection method that can detect individual 15-nm-radius polystyrene nanoparticles, 10-nm gold nanoparticles and influenza A virions in air, and 30 nm polystyrene nanoparticles in water. Our approach relies on measuring changes in the beat note that is produced when an ultra-narrow emission line from a whispering gallery mode microlaser is split into two modes by a nanoscale object, and these two modes then interfere. The ultimate detection limit is set by the laser linewidth, which can be made much narrower than the resonance linewidth of any passive resonator. This means that microlaser sensors have the potential to detect objects that are too small to be detected by passive resonator sensors.