Folding and unfolding kinetics of DNA hairpins in flowing solution by multiparameter fluorescence correlation spectroscopy

Dynamic equilibrium between the folded and unfolded conformations of single stranded DNA hairpin molecules containing polythymine hairpin loops was investigated using simultaneous two-beam fluorescence cross-correlation spectroscopy and single beam autocorrelation spectroscopy. The hairpins were end-labeled with a fluorescent dye and a quencher, such that folding and unfolding of the DNA hairpin primary structure caused the dye fluorescence to fluctuate on the same characteristic time scale as the folding and unfolding reaction. These fluctuations were observed as the molecules flowed sequentially between two spatially offset, microscopic detection volumes. Cross-correlation analysis of fluorescence from the two detection volumes revealed the translational diffusion and flow properties of the hairpins, as well as the average molecular occupancy of the two volumes. Autocorrelation analysis of the fluorescence from the individual detection volumes revealed the kinetics of hairpin folding and unfolding, with the parameters relating to diffusion, flow, and molecular occupancy constrained to the values determined from the cross-correlation analysis. This allowed unambiguous characterization of the folding and unfolding kinetics, without the need to determine the hydrodynamic properties by analyzing a separate control sample. The analysis revealed nonexponential relaxation kinetics and DNA size-dependent folding times characteristic of dynamic heterogeneity in the DNA hairpin-forming mechanism.     

Bio-assay based on single molecule fluorescence detection in microfluidic channels

A rapid bioassay is described based on the detection of colocalized fluorescent DNA probes bound to DNA targets in a pressure-driven solution flowing through a planar microfluidic channel. By employing total internal reflection excitation of the fluorescent probes and illumination of almost the entire flow channel, single fluorescent molecules can be efficiently detected leading to the rapid analysis of nearly the entire solution flowed through the device. Cross-correlation between images obtained from two spectrally distinct probes is used to determine the target concentration and efficiently reduces the number of false positives. The rapid analysis of DNA targets in the low pM range in less than a minute is demonstrated.     

Microfluidic nanoplasmonic-enabled device for multiplex DNA detection

We describe a rapid, quantitative, multiplex, self-labelled, and real-time DNA biosensor employing Ag nanoparticle-bound DNA hairpin probes immobilized in a microfluidic channel. Capture of complementary target DNAs by the microarrayed DNA hairpin probes results in a positive fluorescence signal via a conformational change of the probe molecules, signalling the presence of target DNAs. The device's capability for quantitative analyses was evaluated and a detection time as low as 6 min (with a target flow rate of 0.5 μl min(-1)) was sufficient to generate significant detection signals. This detection time translates to merely 3 μl of target solution consumption. An unoptimized sensitivity of 500 pM was demonstrated for this device.     

Controlled Logic Gates—Switch Gate and Fredkin Gate Based on Enzyme‐Biocatalyzed Reactions Realized in Flow Cells

Controlled logic gates, where the logic operations on the Data inputs are performed in the way determined by the Control signal, were designed in a chemical fashion. Specifically, the systems where the Data output signals directed to various output channels depending on the logic value of the Control input signal have been designed based on enzyme biocatalyzed reactions performed in a multi‐cell flow system. In the Switch gate one Data signal was directed to one of two possible output channels depending on the logic value of the Control input signal. In the reversible Fredkin gate the routing of two Data signals between two output channels is controlled by the third Control signal. The flow devices were created using a network of flow cells, each modified with one enzyme that biocatalyzed one chemical reaction. The enzymatic cascade was realized by moving the solution from one reacting cell to another which were organized in a specific network. The modular design of the enzyme‐based systems realized in the flow device allowed easy reconfiguration of the logic system, thus allowing simple extension of the logic operation from the 2‐input/3‐output channels in the Switch gate to the 3‐input/3‐output channels in the Fredkin gate. Further increase of the system complexity for realization of various logic processes is feasible with the use of the flow cell modular design.     

Six orders of magnitude dynamic range in capillary electrophoresis with ultrasensitive laser-induced fluorescence detection

An ultrasensitive laser-induced fluorescence detector was used with capillary electrophoresis for the study of 5-carboxy-tetramethylrhodamine. The raw signal from the detector provided roughly three orders of magnitude dynamic range. The signal saturated at high analyte concentrations due to the dead time associated with the single-photon counting avalanche photodiode employed in the detector. The signal can be corrected for the detector dead time, providing an additional order of magnitude dynamic range. To further increase dynamic range, two fiber-optic beam-splitters were cascaded to generate a primary signal and two attenuated signals, each monitored by a single-photon counting avalanche photodiode. The combined signals from the three photodiodes are reasonably linear from the concentration detection limit of 3 pM to 10 μM, the maximum concentration investigated, a range of 3,000,000. Mass detection limits were 150 yoctomoles injected onto the capillary.     

Effect of the Solvent Flow Rate on the Ionization Efficiency in Atmospheric Pressure Photoionization-Mass Spectrometry

In the novel atmospheric pressure photoionization-mass spectrometry the ionization efficiency has been observed to decrease when the solvent flow rate is increased. The effect of the flow rate on the ionization efficiency was studied by comparing the behavior of two analytes, one of which is ionized through charge exchange, the other through proton transfer. Additional information about the ion loss mechanisms was obtained by comparing results obtained with two different APPI ion sources: a Sciex prototype and the Agilent/Syagen APPI source. In addition to the measurements done by using the mass analyzer, the total ion current in the ion source was obtained by measuring the currents of the ions arriving at curtain/end plate and orifice/capillary of the two mass spectrometers. The total ion current measurements showed a significant decrease at high solvent flow rates. Loss of dopant radical cations was thought to be the reason for the signal decrease of the analytes formed through charge exchange. Analytes formed through proton transfer were not as seriously ected by the high solvent flow rates, but some saturation of their signal was nevertheless observed. Loss of photons through absorption by solvent vapor is another mechanism that can be held responsible for a reduction of the total number of ions produced by the APPI source.     

Bioelectronic Interface Connecting Reversible Logic Gates Based on Enzyme and DNA Reactions

It is believed that connecting biomolecular computation elements in complex networks of communicating molecules may eventually lead to a biocomputer that can be used for diagnostics and/or the cure of physiological and genetic disorders. Here, a bioelectronic interface based on biomolecule‐modified electrodes has been designed to bridge reversible enzymatic logic gates with reversible DNA‐based logic gates. The enzyme‐based Fredkin gate with three input and three output signals was connected to the DNA‐based Feynman gate with two input and two output signals—both representing logically reversible computing elements. In the reversible Fredkin gate, the routing of two data signals between two output channels was controlled by the control signal (third channel). The two data output signals generated by the Fredkin gate were directed toward two electrochemical flow cells, responding to the output signals by releasing DNA molecules that serve as the input signals for the next Feynman logic gate based on the DNA reacting cascade, producing, in turn, two final output signals. The Feynman gate operated as the controlled NOT gate (CNOT), where one of the input channels controlled a NOT operation on another channel. Both logic gates represented a highly sophisticated combination of input‐controlled signal‐routing logic operations, resulting in redirecting chemical signals in different channels and performing orchestrated computing processes. The biomolecular reaction cascade responsible for the signal processing was realized by moving the solution from one reacting cell to another, including the reacting flow cells and electrochemical flow cells, which were organized in a specific network mimicking electronic computing circuitries. The designed system represents the first example of high complexity biocomputing processes integrating enzyme and DNA reactions and performing logically reversible signal processing.     

Evaluation of noisy FIAS using the cross-covariance function

Summary Cross-correlation between FIA-signals and a sequence of rectangular signals reduces analytical noise and improves the limit of decision. This is pointed out by means of simulations and by FIA-AAS.     

Evaluation of transversal and longitudinal dispersion in a flow injection system by exploiting laser induced fluorescence: influence of flow-cell positioning

A fully mechanized set-up was built for the experimental determination of bi-dimensional dispersion with high spatial resolution (2400 μm²). Gravitational and wall effects in a single stream were evaluated by using time-based sampling and a micro-flow cell. Vertical upward and downward flows as well as horizontal flows were investigated. Ethylene glycol (MEG) and Rhodamine B in MEG were used as carrier and sample solutions, respectively. Longitudinal profiles were obtained by laser induced total fluorescence (LIF) at up to 19 transversal sites and combined to generate high-resolution bi-dimensional profiles. A two frontal maxima pattern was observed for all flows. The volumetric fraction of RB shape was highly stretched for downward flow and there was high asymmetry for horizontal flow. The sensitivity of three dispersion parameters was evaluated: maximum peak value, peak half-width at half-height, and peak area.Data modeling showed that the tanks-in-series was more sensitive to wall effects, had good adjustment with only one tank for upward and horizontal flow and needed two tanks for downward flow which was attributed to the latter having higher dispersion. A black box empirical modeling described better the gravitational effect and allowed to identify a parameter sensitive to upward and downward flow as well as hinting to two inner streams within the horizontal flow. It also pointed to a wall dispersion contribution of twice that of the liquid-liquid dispersion.     

An absorbance-based micro-fluidic sensor for diffusion coefficient and molar mass determinations

The H-Sensor reported herein is a micro-fluidic device compatible with flow injection analysis (FIA) and high performance liquid chromatography (HPLC). The device detects analytes at two separate off-chip absorbance flow cells, providing two simultaneous absorbance measurements. The ratio of these two absorbance signals contains analyte diffusion coefficient information. A theoretical model for the sensing mechanism is presented. The model relates the signal Ratio to analyte diffusion coefficient. The model is qualitatively evaluated by comparing theoretical and experimental signal Ratio values. Experimental signal Ratios were collected via FIA for a variety of analytes, including sodium azide, benzoic acid, amino acids, peptides, and proteins. Measuring absorbance at multiple wavelengths provides higher order data allowing the analyte signals from mixtures to be deconvolved via classical least squares (CLS). As a result of the H-Sensor providing two simultaneous signals as a function of time for each sample injection, two simulated second-order HPLC chromatograms were generated using experimental H-Sensor data. The chemometric deconvolution method referred to as the generalized rank annihilation method (GRAM) was used to demonstrate chromatographic and spectroscopic deconvolution. GRAM also provides the signal Ratio value, therefore simultaneously obtaining the analyte diffusion coefficient information during deconvolution. The two chromatograms successfully serve as the standard and unknown for the GRAM deconvolution. GRAM was evaluated on chromatograms at various chromatographic resolutions. GRAM was found to function to a chromatographic resolution at and above 0.25 with a percent quantitative error of less then 10%.     

Multiple electrosprays generated from a single polycarbonate microstructured fibre

Electrospray ionization (ESI) has been invaluable to the mass spectrometric detection of biomolecules, due largely to the sensitivity afforded by the ionization technique. Lower flow rates, e.g. in the nanoelectrospray regime, result in smaller initial electrosprayed droplets, leading to higher ionization efficiency and greater signal. One approach to improving sensitivity without lowering flow rate is to generate multiple electrosprays (MESs) from the same sample, essentially splitting one larger flow into smaller flows in the nanoESI regime. Presented here is a series of novel MES emitters in the form of polycarbonate fibres. Based on microstructured fibre (MSF) technology whereby a set of homogeneous parallel channels are formed in a heat‐drawn fibre intended to conduct light, a custom design was fabricated in which 3, 6, 9 and 12 holes were arranged in a radial pattern to prevent inhomogeneities in the electric field. The MSFs have dimensions that are compatible with current standards in nanoESI equipment, and the tip is more compatible with standard MS orifices than other larger multielectrospray emitters. By measuring the spray current provided by the various emitters under the same solvent/voltage/total flow rate conditions, a plot was obtained clearly demonstrating the expected dependence on the square root of the number of holes, i.e. the number of independent electrosprays. With this firm proof of principle using this design/format, further effort is justified in developing similar emitters in alternative materials that better prevent surface wetting and allow greater hole density, ultimately leading to greater signal enhancement. Copyright © 2012 John Wiley & Sons, Ltd.     

In situ electrochemical ESR and voltammetric studies on the anodic oxidation of para-haloanilines in acetonitrile

An in situ electrochemical electron spin resonance (ESR) study on the electro-oxidation of para-chloroaniline, para-bromoaniline, and para-iodoaniline dissolved in acetonitrile at gold electrodes is reported. ESR spectra obtained using a tubular flow cell reveal the presence of a paramagnetic dimer product derived from para-aminodiphenylamine, during oxidative electrolysis, suggesting the coupling of reactive electrogenerated radical cations with neutral parent haloaniline molecules. The ESR signal intensity behaves in a manner expected for a radical species reacting with second-order kinetics, suggesting the paramagnetic dimer is, itself, unstable. The theory describing the ESR signal intensity flow rate behavior for this reaction mechanism is developed for the tubular arrangement and used to extract mechanistic and kinetic data from the experimental results for the cases of para-chloroaniline and para-bromoaniline. Further mechanistic aspects, including proton and halide ion expulsion during dimerization, are explored voltammetrically and with the aid of digital simulations using Digisim. Comparison of the ESR signal and voltammetric measurements suggests that an additional mechanism operates which does not lead to paramagnetic products. Additionally, the in situ electrolysis of N,N-dimethyl-para-bromoaniline is reported to generate the stable radical cation of N,N,N',N'-tetramethylbenzidine, and a mechanism of electro-oxidation is, thus, proposed.     

Intermolecular communication on a liposomal membrane: enzymatic amplification of a photonic signal with a gemini peptide lipid as a membrane-bound artificial receptor

A supramolecular system that can activate an enzyme through photo-isomerization was constructed by using a liposomal membrane scaffold. The design of the system was inspired by natural signal transduction systems, in which enzymes amplify external signals to control signal transduction pathways. The liposomal membrane, which provided a scaffold for the system, was prepared by self-assembly of a photoresponsive receptor and a cationic synthetic lipid. NADH-dependent L-lactate dehydrogenase, the signal amplifier, was immobilized on the liposomal surface by electrostatic interactions. Recognition of photonic signals by the membrane-bound receptor induced photo-isomerization, which significantly altered the receptor's metal-binding affinity. The response to the photonic signal was transmitted to the enzyme by Cu(2+) ions. The enzyme amplified the chemical information through a catalytic reaction to generate the intended output signal.     

Potential controlling highly-efficient catalysis of wheat-like silver particles for electrochemiluminescence immunosensor labeled by nano-Pt@Ru and multi-sites biotin/streptavidin affinity

The potential controlling silver catalysis for Ru(bpy)(3)(2+) electrochemiluminescence (ECL) signal at a special potential -0.4～1.25 V was newly developed as the new ECL signal amplification strategy for ultrasensitive protein detection. Firstly, the wheat-like deposited silver (DpAg) particles were modified on the bare glass carbon electrode (GCE) surface by cyclic voltammetry deposition to capture the primary antibodies and then bind the antigen analytes. Secondly, as a sandwich immunoreaction format, the secondary antibodies conjugated with the Ru(bpy)(3)(2+)-doped Pt (Pt@Ru) nanoparticles by the multi-sites biotin/streptavidin (SA) affinity can be captured onto the electrode surface to generate ECL signal. In the proposed Ru(bpy)(3)(2+) ECL system without any co-reactant, the detected ECL signal was amplified due to following multiple amplification strategies: (1) the ECL catalysis for Ru(bpy)(3)(2+) was performed by electro-inducing the DpAg particles to generate Ag(+) ion and controlled by the special potential. The catalyzer Ag(+) was produced near the electrode surface and reproduced by cyclic potential scan, which improved the catalytic efficiency. (2) The amount of the ECL signal probes linked to secondary antibodies were amplified by the adsorption of Pt nanoparticles and the multiple sites bridge linkage of biotin/SA. These new multiple signal amplification strategies made the proposed ECL immunosensor achieve ultrasensitive detection for model protein human IgG with a detection limit down to 3 pg mL(-1), which can be further extended to the detection of disease biomarkers.     

Intermolecular Communication on a Liposomal Membrane: Enzymatic Amplification of a Photonic Signal with a Gemini Peptide Lipid as a Membrane‐Bound Artificial Receptor

A supramolecular system that can activate an enzyme through photo‐isomerization was constructed by using a liposomal membrane scaffold. The design of the system was inspired by natural signal transduction systems, in which enzymes amplify external signals to control signal transduction pathways. The liposomal membrane, which provided a scaffold for the system, was prepared by self‐assembly of a photoresponsive receptor and a cationic synthetic lipid. NADH‐dependent L ‐lactate dehydrogenase, the signal amplifier, was immobilized on the liposomal surface by electrostatic interactions. Recognition of photonic signals by the membrane‐bound receptor induced photo‐isomerization, which significantly altered the receptor’s metal‐binding affinity. The response to the photonic signal was transmitted to the enzyme by Cu²⁺ ions. The enzyme amplified the chemical information through a catalytic reaction to generate the intended output signal.     

Cross-correlation masks for automatic detection of spectral information

Cross-correlation masks based on spectral intensities and binary spectral peak positions are evaluated for detection and quantitation of atomic emission spectra. The inductively coupled plasma emission spectra of nickel and vanadium as acquired with a photodiode array spectrometer were utilized to test and illustrate the cross-correlation procedures.     

A microfluidic flow distributor generating stepwise concentrations for high-throughput biochemical processing

In this paper, we describe a microfluidic device in which solutions with stepwise concentrations can be accurately generated by continuously introducing two kinds of miscible liquids from each inlet, and biochemical processing can be conducted at the various conditions. Introduced liquid flows are geometrically divided into a number of downstream flows through multiple distribution channels, and each divided flow is then mixed with the divided flow of another liquid at a confluent point. The lengths of the precisely designed distribution channels determine the mixing ratio of the two liquids, without the influence of flow rate. In this study, a PDMS microfluidic device able to generate nine different concentrations was fabricated, and the performance of this device was estimated via colorimetric assay. As a biological application of this device, cell cultivation was performed under different concentration conditions. Due to its simplicity of operation, this microfluidic flow distributor will be applied to various kinds of biological analysis and screening systems.     

Switching of the enzymatic activity synchronized with signal recognition by an artificial DNA receptor on a liposomal membrane

We constructed a supramolecular system on a liposomal membrane that is capable of activating an enzyme via DNA hybridization. The design of the system was inspired by natural signal transduction systems, in which enzymes amplify external signals to control signal transduction pathways. The liposomal membrane, providing a platform for the system, was prepared by the self-assembly of an oligonucleotide lipid, a phospholipid and a cationic synthetic lipid. The enzyme was immobilized on the liposomal surface through electrostatic interactions. Selective recognition of DNA signals was achieved by hybridizing the DNA signals with the oligonucleotide lipid embedded in the liposome. The hybridized DNA signal was sent to the enzyme by a copper ion acting as a mediator species. The enzyme then amplified the event by the catalytic reaction to generate the output signal. In addition, our system demonstrated potential for the discrimination of single nucleotide polymorphisms.     

Mixing in microchannels based on hydrodynamic focusing and time-interleaved segmentation: modelling and experiment

This paper theoretically and experimentally investigates a micromixer based on combined hydrodynamic focusing and time-interleaved segmentation. Both hydrodynamic focusing and time-interleaved segmentation are used in the present study to reduce mixing path, to shorten mixing time, and to enhance mixing quality. While hydrodynamic focusing reduces the transversal mixing path, time-interleaved sequential segmentation shortens the axial mixing path. With the same viscosity in the different streams, the focused width can be adjusted by the flow rate ratio. The axial mixing path or the segment length can be controlled by the switching frequency and the mean velocity of the flow. Mixing ratio can be controlled by both flow rate ratio and pulse width modulation of the switching signal. This paper first presents a time-dependent two-dimensional analytical model for the mixing concept. The model considers an arbitrary mixing ratio between solute and solvent as well as the axial Taylor-Aris dispersion. A micromixer was designed and fabricated based on lamination of four polymer layers. The layers were machined using a CO2 laser. Time-interleaved segmentation was realized by two piezoelectric valves. The sheath streams for hydrodynamic focusing are introduced through the other two inlets. A special measurement set-up was designed with synchronization of the mixer's switching signal and the camera's trigger signal. The set-up allows a relatively slow and low-resolution CCD camera to freeze and to capture a large transient concentration field. The concentration profile along the mixing channel agrees qualitatively well with the analytical model. The analytical model and the device promise to be suitable tools for studying Taylor-Aris dispersion near the entrance of a flat microchannel.