The fluctuating enzyme: a single molecule approach

The excess of structural degrees of freedom in a protein enzyme opens questions about the conformational homogeneity. We studied single horseradish peroxidase enzyme turnovers by fluorescence spectroscopy. Application of a two-state dynamic model to the measured data shows exponential product dissociation kinetics, but a large distribution of rates for the enzyme to form the enzyme-product complex. The experiments show that in addition to the peroxidative cycle thermodynamic fluctuation phenomena on a wide range of time scales affect enzyme activity.     

Beyond power laws: a new approach for analyzing single molecule photoluminescence intermittency

The photoluminescence intermittency (PI) exhibited by single emitters has been studied for over a decade. To date, the vast majority of PI analyses involve parsing the data into emissive and non-emissive events, constructing histograms of event durations, and fitting these histograms to either exponential or power law probability distributions functions (PDFs). Here, a new method for analyzing PI data is presented where the data are used directly to construct a cumulative distribution function (CDF), and maximum-likelihood estimation techniques are used to determine the best fit of a model PDF to the CDF. Statistical tests are then employed to quantitatively evaluate the hypothesis that the CDF (data) is represented by the model PDF. The analysis method is outlined and applied to PI exhibited by single CdSe∕CdS core-shell nanocrystals and the organic chromophore violamine R isolated in single crystals of potassium-acid phthalate. Contrary to previous studies, the analysis presented here demonstrates that the PI exhibited by these systems is not described by a power law. The analysis developed here is also used to quantify heterogeneity within PI data obtained from a collection of CdSe/CdS nanocrytals, and for the determination of statistically significant changes in PI accompanying perturbation of the emitter. In summary, the analysis methodology presented here provides a more statistically robust approach for analyzing PI data.     

High-throughput single molecule screening of DNA and proteins

We report a novel imaging technology for real time comprehensive analysis of molecular alterations in cells and tissues appropriate for automation and adaptation to high-throughput applications. With these techniques it should eventually be possible to perform simultaneous analysis of the entire contents of individual biological cells with a sensitivity and selectivity sufficient to determine the presence or absence of a single copy of a targeted analyte (e.g., DNA region, RNA region, protein), and to do so at a relatively low cost. The technology is suitable for DNA and RNA through sizing or through fluorescent hybridization probes, and for proteins and small molecules through fluorescence immunoassays. This combination of the lowest possible detection limit and the broadest applicability to biomolecules represents the final frontier in bioanalysis. The general scheme is based on novel concepts for single molecule detection (SMD) and characterization recently demonstrated in our laboratory. Since minimal manipulation is involved, it should be possible to screen large numbers of cells in a short time to facilitate practical applications. This opens up the possibility of finding single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification.     

Probing redox proteins on a gold surface by single molecule fluorescence spectroscopy

The interaction between the fluorescently labeled redox protein, azurin, and a thin gold film is characterized using single-molecule fluorescence intensity and lifetime measurements. Fluorescence quenching starts at distances below 2.3 nm from the gold surface. At shorter distances the quantum yield may decrease down to fourfold for direct attachment of the protein to bare gold. Outside of the quenching range, up to fivefold enhancement of the fluorescence is observed on average with increasing roughness of the gold layer. Fluorescence-detected redox activity of individual azurin molecules, with a lifetime switching ratio of 0.4, is demonstrated for the first time close to a gold surface.     

DNA-psoralen interaction: a single molecule experiment

By attaching one end of a single lambda-DNA molecule to a microscope coverslip and the other end to a polystyrene microsphere trapped by an optical tweezers, we can study the entropic elasticity of the lambda-DNA by measuring force versus extension as we stretch the molecule. This powerful method permits single molecule studies. We are particularly interested in the effects of the photosensitive drug psoralen on the elasticity of the DNA molecule. We have illuminated the sample with different light sources, studying how the different wavelengths affect the psoralen-DNA linkage. To do this, we measure the persistence length of individual DNA-psoralen complexes.     

Substrate-mediated intermolecular interactions: a quantitative single molecule analysis

Long-range intermolecular interactions mediated by the surface are believed to be responsible for many effects in surface science, including molecular ordering, formation of nanostructures, and aligning reactive intermediates in catalysis. Here, we use scanning tunneling microscopy to probe the weak substrate-mediated interactions in benzene overlayers on Au{111} at 4 K. Using an automated procedure to monitor single molecule motion, we are able to quantify the substrate-mediated interaction strength. We explain quantitatively both the kinetics of the benzene motion and the thermodynamics that determine the packing structures benzene adopts in this system in light of these substrate-mediated interactions.     

Nanoengineered multiscale hierarchical structures with tailored wetting properties

A simple method for fabricating micro/nanoscale hierarchical structures is presented using a two-step temperature-directed capillary molding technique. This lithographic method involves a sequential application of the molding process in which a uniform polymer-coated surface is molded with a patterned mold by means of capillary force above the glass transition temperature of the polymer. Various microstructures and nanostructures were fabricated with minimum resolution down to approximately 50 nm with good reproducibility. Also contact angle measurements of water indicated that two wetting states coexist on a multiscale hierarchical structure where heterogeneous wetting is dominant for the microstructure and homogeneous wetting for the nanostructure. A simple theoretical model combining these two wetting states was presented, which was in good agreement with the experimental data. Using this approach, multiscale hierarchical structures for biomimetic functional surfaces can be fabricated with precise control over geometrical parameters and the wettability of a solid surface can be tailored in a controllable manner.     

Fabrication of micro reaction cages with tailored properties

Hollow polyelectrolyte capsules in micro- and submicrometer size were prepared. Their interior was functionalized by a "ship in bottle" synthesis of copolymers. While the monomers permeated the capsule wall easily, the formed polymers remained in the capsule cage. The physicochemical properties of the capsule interior such as ion strength, pH, light absorption, and fluorescence could be controlled independently from the surrounding solvent by means of the chemical nature of the captured polymer. In case of polyelectrolytes the osmotic pressure of the counterions led to a swelling of the capsules which can be important for micromechanics. The functionalization with light-sensitive materials allowed selective photoreactions inside the capsules. Synthesis of polyelectrolytes at high concentration resulted in an intertwining of the capsule wall with the polymer. The modified walls behaved like ion exchange membranes and showed selectivity toward adsorption and permeation of organic ions. The modified capsules offer many possibilities for novel applications as containers for controlled precipitation, as nanoreactors for catalyzed reactions, or as sensors.     

Driving the Bloch vector of a single molecule: towards a triggered single photon source

We propose the method of rapid adiabatic passage to prepare a single molecule in its fluorescing excited state. Spontaneous emission from this state gives rise to a single photon. Since the adiabatic passage can be performed on command, the molecule can be used as a triggered single photon source. Preliminary experiments and quantum Monte-Carlo simulations demonstrate the feasibility of this scheme.     

Tandem repeating modular proteins avoid aggregation in single molecule force spectroscopy experiments

We have used single molecule force spectroscopy to explore the unfolding and refolding behavior of the immunoglobulin-like I27 protein in aqueous 2,2,2-trifluoroethanol (TFE). In bulk solution experiments, a 28% v/v TFE solution has previously been observed to enhance intermolecular attractions and lead to misfolding and aggregation of tandem modular proteins of high sequence identity. In our single molecule experiments, however, we measure successful refolding of the polyprotein I27(8) in all TFE solutions up to 35% v/v. Using a single molecule micromanipulation technique, we have shown that refolding of a polyprotein with identical repeats is not hindered by the presence of this cosolvent. These experimental results provide new insight into the properties of tandem repeating proteins and raise interesting questions as to the evolutionary success of such proteins in avoiding misfolding and aggregation.     

Covalent attachment of proteins to calcite surface for the single molecule experiments

Two protocols of covalent attachment of proteins onto the calcite surface, viz. one using the metallochelat and second using the aminohexil, are elaborated. Single molecule force spectroscopy method has been used to test their efficiency and practical applicability. Experiments were performed measuring the specific interaction force between bovine serum albumin (BSA) fixed onto the freshly cleaved calcite single crystal surface (procedure under the study here) and its polyclonal antibody (Ab-BSA) immobilized onto an AFM tip using standard and well studied procedure. We found the conditions, when up to 3–3.5% of tip-sample approaches lead to the formation of a single specific bond.     

Engineering proteins: keeping up with biology

A wide range of biological laboratories have adopted protein engineering techniques, altering the way biochemical research is carried out. Ironically, this broad success has increased the challenges faced by researchers at the chemistry-biology interface.     

Imaging of tautomerism in a single molecule

Fluorescence imaging is used to visualize directly the transfer of two inner hydrogen atoms in single porphycene molecules. This reaction leads to a chemically equivalent but differently oriented structure and hence results in a rotation of the transition dipole moments. By probing single immobilized molecules with an azimuthally polarized laser beam in the focal spot of a confocal microscope we observe ring-like emission patterns, possible only for a chromophore with two nearly orthogonal transition dipole moments. Numerical simulations of the observed emission patterns yield a value of 72 degrees for the angle between the S0-S1 transition moments in the two tautomeric forms.     

Single molecule magnet with single lanthanide ion

The single molecule magnet behavior of metal complexes containing a single magnetic ion was first reported for phthalocyaninato–lanthanide complexes with a “double-decker” structure. In this paper, recent attempts by the author and coworkers at revealing the magnetism and electronic structures of the mononuclear and dinuclear lanthanide complexes with phthalocyaninato ligands are reviewed.     

An amphiphilic molecule with a single fluorophore exhibits multiple stimuli-responsive behavior

Fluorescent materials that respond to multiple stimuli have broad applications ranging from sensing and bioimaging to information encryption. Herein, we report the design and synthesis of a single-fluorophore-based amphiphile DCSO, which shows temperature-, solvent-, humidity-, and radiation-dependent fluorescence. DCSO consists of a dicyanostilbene (DCS) group as a rigid hydrophobic core with oligo(ethylene glycol) (OEG) chains at both ends as a flexible hydrophilic periphery. The DCS group acts as a highly efficient fluorophore, while the OEG chain endows the molecule with thermo-responsiveness. Fluorescent colors can vary from blue to green to yellow in response to external stimuli. On the basis of light radiation, we demonstrate that this system can be applied to time-dependent information encryption, in which the correct information can only be read at a specific time under irradiation. This work further demonstrates the usefulness and application of single-fluorophore-based luminescent materials with multiple stimuli-responsive functions.     

Simultaneous detection of small molecules,proteins and microRNAs using single molecule arrays

Biological samples such as blood, urine, cerebrospinal fluid and saliva contain a large variety of proteins, nucleic acids, and small molecules. These molecules can serve as potential biomarkers of disease and therefore, it is desirable to simultaneously detect multiple biomarkers in one sample. Current detection techniques suffer from various limitations including low analytical sensitivity and complex sample processing. In this work, we present an ultrasensitive method for simultaneous detection of small molecules, proteins and microRNAs using single molecule arrays (Simoa). Dye-encoded beads modified with specific capture probes were used to quantify each analyte. Multiplex competitive Simoa assays were established for simultaneous detection of cortisol and prostaglandin E2. In addition, competitive and sandwich immunoassays were combined with a direct nucleic acid hybridization assay for simultaneous detection of cortisol, interleukin 6 and microRNA 141. The multi-analyte Simoa assay shows high sensitivity and specificity, which provides a powerful tool for the analysis of many different samples.

The first example of multiplexed detection of proteins, nucleic acids, and small molecules using single molecule measurement methodology.     

Thermodynamic, structural, and nanomechanical properties of a fluorous biphasic material

The dynamics of the amphiphilic semifluorinated F(CF2)12(CH2)12H (F12H12) alkane that undergoes two condensed phase transitions have been investigated by Brillouin light spectroscopy, shear rheometry, small- (SAXS) and wide-angle (WAXS) X-ray scattering, and thermodynamic PVT measurements. The solid (I)-solid (II) transition (Ts) is marked by a stronger temperature dependence of the sound velocity in phase II and by a 2 orders of magnitude drop of the shear modulus. Between the Ts and the melting transition (Tm), the presence of two phonons implies a coexistence of solid (II) and amorphous (liquid) regions in the submicrometer range at thermal equilibrium as revealed by the SAXS pattern of a single reflection superimposed on a very broad amorphous halo. This intriguing finding of a transient, very slow (over 10 h) solid/liquid coexistence within phase II is rationalized by a two-stage mechanism for melting of the smectic phase (II) of F12H12. A refinement of the known packing motifs for the two solid-state structures is proposed.