Shedding light on biomolecule conformational dynamics using fluorescence measurements of trapped ions

Biomolecule conformational change has been widely investigated in solution using several methods; however, much less experimental data about structural changes are available for completely isolated, gas-phase biomolecules. Studies of conformational change in unsolvated biomolecules are required to complement the interpretation of mass spectrometry measurements and in addition, can provide a means to directly test theoretical simulations of biomolecule structure and dynamics independent of a simulated solvent. In this Feature Article, we review our recent introduction of a fluorescence-based method for probing local conformational dynamics in unsolvated biomolecules through interactions of an attached dye with tryptophan (Trp) residues and fields originating on charge sites. Dye-derivatized biomolecule ions are formed by electrospray ionization and are trapped in a variable-temperature quadrupole ion trap in which they are irradiated with either continuous or short pulse lasers to excite fluorescence. Fluorescence is measured as a function of temperature for different charge states. Optical measurements of the dye fluorescence include average intensity changes, changes in the emission spectrum, and time-resolved measurements of the fluorescence decay. These measurements have been applied to the miniprotein, Trp-cage, polyproline peptides and to a beta-hairpin-forming peptide, and the results are presented as examples of the broad applicability and utility of these methods. Model fits to Trp-cage fluorescence data measured as a function of temperature provide quantitative information on the thermodynamics of conformational changes, which are reproduced well by molecular dynamics. Time-resolved measurements of the fluorescence decays of Trp-cage and small polyproline peptides definitively demonstrate the occurrence of fluorescence quenching by the amino acid Trp in unsolvated biomolecules.     

Effect of buffer gas on the fluorescence yield of trapped gas-phase ions

We investigated the dependence of three different gases, helium, argon, and nitrogen, on the fluorescence signal intensity of rhodamine 6G cations in the gas phase. The method is based on laser-induced fluorescence of ions trapped in a Fourier transform ion cyclotron mass spectrometer. We found that the use of helium results in the highest fluorescence signal, while no fluorescence was detected when using argon under the same conditions.     

Photodissociation of trapped ions

The ion-trapping ion cyclotron resonance spectrometer, or Fourier transform mass spectrometer, provides a powerful and convenient environment for the study of photodissociation of gas-phase ions. This capability has been explored for about 30 years in a number of laboratories including our own. A variety of developments and applications, historical and current, are organized here under five broad headings: (1) optical spectroscopy of ions; (2) kinetics of the dissociation process; (3) dynamics of the dissociation process; (4) thermochemistry of dissociation; and (5) probing the structure and energy of the ions.     

Electronic fluorescence spectra of gas-phase positive molecular ions

A new technique for observing electronic spectra of gas-phase positive molecular ions is described. The ions are produced by electron impact on a supersonic molecular beam, and the resulting fluorescent radiation is dispersed. Some results on N₂, N₂O and CO₂ are presented. They suggest that this technique could be most productive for studying large polyatomic cations.     

Infrared spectroscopy of cold trapped molecular ions using He‐tagging

This review summarizes experimental activities to study the structure of molecular ions via He tagging. The method is based on the attachment of a weakly bound helium atom to a cold ion followed by laser‐induced predissociation (LIP). Since my early involvements (it started in 1977 with a letter from Y.T. Lee), radio frequency (rf) ion traps and ion guides have been important elements in instruments dedicated to ion spectroscopy. Accumulating ions in a ring electrode trap (RET) and confining them together with the laser‐induced photofragments in a long octopole has been demonstrated in 1978 in Berkeley via photodissociation of metastable O₂⁺ ions. In the early stage of this instrument, as well as in various further developments, supersonic expansions have been used to create weakly bound complexes. An important step forward for ion spectroscopy was to push the conditions of cryogenic ion traps so far that, finally, He atoms could be attached to almost any mass‐selected ion of interest, including multiply charged ions and C₆₀⁺. Currently, modern ion storage instruments reach temperatures below 3 K and can be operated at helium densities above 10¹⁶ cm⁻³, opening up many avenues of application in spectroscopy, reaction dynamics, and analytical chemistry. In addition to a personal historical review, I discuss recent progress made with new cryogenic ion traps, especially in the field of He tagging. He‐M⁺ ions have been formed via ternary association for all kind of M⁺ ions ranging from atoms such as He⁺, N⁺, or Fe⁺ via molecules N₂⁺, VO⁺, and H₃⁺ to various polyatomic ions. The in situ synthesis of tagged ions made unique discoveries possible, such as determining the structure of doubly charged benzene, the first identification of a carrier of diffuse interstellar bands, or the characterization of the fundamental 4 electron 4 center system He–H₃⁺. In the conclusions, hints to additional applications will be given, emphasizing on the versatility of temperature‐variable ion traps.     

Antigen detection based on background fluorescence quenching immunochromatographic assay

Gold immunochromatographic assay (GICA) has been around for quite a while, but it is qualitative in the vast majority of applications. A fast, simple and quantitative GICA is in call for better medicine. In the current study, we have established a novel, quantitative GICA based on fluorescence quenching and nitrocellulose membrane background signals, called background fluorescence quenching immunochromatographic assay (bFQICA). Using model analyte alpha-fetoprotein (AFP), the present study assessed the performance of bFQICA in numerous assay aspects. With serial dilutions of the international AFP standard, standard curves for the calculation of AFP concentration were successfully established. At 10 and 100 ng mL⁻¹ of the international AFP standard, the assay variability was defined with a coefficient of variance at 10.4% and 15.2%, respectively. For samples with extended range of AFP levels, bFQICA was able to detect AFP at as low as 1 ng mL⁻¹. Fluorescence in bFQICA strips stayed constant over months. A good correlation between the results from bFQICA and from a well-established Roche electrochemiluminescence immunoassay was observed in 27 serum samples (r = 0.98, p < 0.001). In conclusion, our study has demonstrated distinctive features of bFQICA over conventional GICA, including utilization of a unique fluorescence ratio between nitrocellulose membrane background and specific signals (F1/F2) to ensure accurate measurements, combined qualitative and quantitative capabilities, and exceptionally high sensitivity for detection of very low levels of antigens. All of these features could make bFQICA attractive as a model for antigen-antibody complex based GICA, and could promote bFQICA to a broad range of applications for investigation of a variety of diseases.     

Complete suppression of the fluorophore fluorescence by combined effect of multiple fluorescence quenching groups: A fluorescent sensor for Cu with zero background signals

The reaction-based fluorescent sensors have attracted increasing attention in the past decades. However, the application of these sensors for accurate sensing was significantly retarded by the background fluorescence from the sensors themselves. In this work, we demonstrated a novel strategy that the background fluorescence of the sensor could be completely eliminated by the combined effect of multiple fluorescence quenching groups. Based on this new strategy, as proof-of-principle study, a fluorescent sensor (CuFS) for Cu²⁺ was judiciously developed. In CuFS, three types of fluorescence quenching groups were directly tethered to a commonly used coumarin fluorophore. The fluorescence of coumarin fluorophore in CuFS was completely suppressed by the combined effect of these fluorescence quenching groups. Upon treatment with 22 μM Cu²⁺, sensor CuFS achieved a dramatic fluorescence enhancement (fluorescence intensity enhanced up to 811-fold) centered at 469 nm. The detection limits was determined to be 12.3 nM. The fluorescence intensity enhancement also showed a good linearity with the Cu²⁺ concentration in the range of 12.3 nM to 2 μM. By fabricating test strips, sensor CuFS can be utilized as a simple tool to detect Cu²⁺ in water samples. Furthermore, the fluorescent sensor was successfully applied in detecting different concentration of Cu²⁺ in living cells.     

A new total antioxidant potential measurements using RP-HPLC assay with fluorescence detection

In this paper, an improved total antioxidant potential (TAP) estimation using high-performance liquid chromatographic (HPLC) assay with fluorometric detection has been described. The principle of this method is based on the hydroxyl radicals generated in the Fenton-like reaction and subsequently detected using hydroxyterephthalic acid (HTPA), which is a reaction product of hydroxyl radicals and terephthalic acid (TPA), working as a sensing compound. HTPA quantity in the samples was measured by fluorescence detector working at excitation and emission wavelengths equal to 312 and 428 nm, respectively. A number of key experimental conditions including the influence of the reaction (incubation) time on the surface areas of HTPA peaks, concentration of Fe(II) ions as well as the influence of concentration of TPA on the surface area of the chromatographic peak of HTPA were optimized to the characteristic feature of TAP measurements. The elaborated assay has been used to evaluate TAP values of selected low-molecular mass compounds like pyrogallol, tryptamine, and n-alcohols (methanol, ethanol, and n-propanol) as well as chlorogenic and ascorbic acids and benzoic acid derivatives, which are commonly present in the food samples.     

Enhanced fluorescence detection of metal ions using light-harvesting mesoporous organosilica

Enhanced fluorescence detection of metal ions was realized in a system consisting of a fluorescent 2,2′‐bipyridine (BPy) receptor and light‐harvesting periodic mesoporous organosilica (PMO). The fluorescent BPy receptor with two silyl groups was synthesized and covalently attached to the pore walls of biphenyl (Bp)‐bridged PMO powder. The fluorescence intensity from the BPy receptor was significantly enhanced by the light‐harvesting property of Bp‐PMO, that is, the energy funneling into the BPy receptor from a large number of Bp groups in the PMO framework which absorbed UV light effectively. The enhanced emission of the BPy receptor was quenched upon the addition of a low concentration of Cu²⁺ (0.15–1.2×10^?6 M ), resulting in the sensitive detection of Cu²⁺. Upon titration of Zn²⁺ (0.3–6.0×10^?6 M ), the fluorescence excitation spectrum was systematically changed with an isosbestic point at 375 nm through 1:1 complexation of BPy and Zn²⁺ similar to that observed in BPy‐based solutions, indicating almost complete preservation of the binding property of the BPy receptor despite covalent fixing on the solid surface. These results demonstrate that light‐harvesting PMOs have great potential as supporting materials for enhanced fluorescence chemosensors.     

Amplified fluorescence detection of mercury(II) ions (Hg2+) using target-induced DNAzyme cascade with catalytic and molecular beacons

In this work, a fluorescent sensing strategy was developed for the detection of mercury(II) ions (Hg(2+)) in aqueous solution with excellent sensitivity and selectivity using a target-induced DNAzyme cascade with catalytic and molecular beacons (CAMB). In order to construct the biosensor, a Mg(2+)-dependent DNAzyme was elaborately designed and artificially split into two separate oligonucleotide fragments. In the presence of Hg(2+), the specific thymine-Hg(2+)-thymine (T-Hg(2+)-T) interaction induced the two fragments to produce the activated Mg(2+)-dependent DNAzyme, which would hybridize with a hairpin-structured MB substrate to form the CAMB system. Eventually, each target-induced activated DNAzyme could catalyze the cleavage of many MB substrates through true enzymatic multiple turnovers. This would significantly enhance the sensitivity of the Hg(2+) sensing system and push the detection limit down to 0.2 nM within a 20 min assay time, much lower than those of most previously reported fluorescence assays. Owning to the strong coordination of Hg(2+) to the T-T mismatched pairs, this proposed sensing system exhibited excellent selectivity for Hg(2+) detection, even in the presence of 100 times of other interferential metal ions. Furthermore, the applicability of the biosensor for Hg(2+) detection in river water samples was demonstrated with satisfactory results. These advantages endow the sensing strategy with a great potential for the simple, rapid, sensitive, and specific detection of Hg(2+) from a wide range of real samples.     

Laser-induced fluorescence of trapped gas-phase molecular ions generated by internal source matrix-assisted laser desorption/ionization in a Fourier transform ion cyclotron resonance mass spectrometer

The combination of laser-induced fluorescence with mass spectrometry opens up new possibilities both for detection purposes and for structural studies of trapped biomolecular ions in the gas phase. However, this approach is experimentally very challenging, and only a handful of studies have been reported so far. In this contribution, a novel scheme for laser-induced fluorescence measurements of ions trapped inside a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer will be introduced. It is based on an open FT-ICR cell design, continuous wave axial excitation of the fluorescence, orthogonal photon collection by fiber optics, and single photon counting detection. Rhodamine 6G ions generated by an internal matrix-assisted laser desorption/ionization source were used to develop and test the set-up. Due to photobleaching processes, the excitation laser power and the observation time window have to be carefully optimized. An ion tomography method was used to align the excitation laser. Potential applications for studying the gas-phase structure of fluorescent biomolecular ions and for investigating fluorescence resonance energy transfer of donor-acceptor pairs will be presented.     

A new strategy for ratiometric fluorescence detection of transition metal ions

A novel design strategy for ratiometric fluorescence signaling of transition metal ions, involving both photoinduced electron transfer and resonance energy transfer mechanisms, has been tested on a model system comprising dual fluorophores.     

Aptamer-based fluorescence sensor for rapid detection of potassium ions in urine

We unveil a new homogeneous assay-using OliGreen and an ATP-binding aptamer-for the highly selective and sensitive detection of potassium ions.     

Synthesis of benzanthron derivatives for selective detection by fluorescence of copper ions

The paper reports on the synthesis of two new benzanthron derivatives with intense yellow-orange fluorescence and their copolymers with styrene. The photophysical characteristics of the new low and high molecular weight fluorescent compounds have been studied in organic solvents of different polarity. The effect of the chemical nature of the C-3 substituent on the photophysical properties of the new dyes is discussed. The complexes formed between the benzanthron fluorophores and metal cations in solution have been studied with regard to potential applications as fluorescent sensors for metal ion contamination. The results show that the C-3 substituent determines a selective response to the presence of Cu²⁺ cations. In the case of copolymers the effect of the metal cations on the fluorescence intensity is negligible.     

Direct molecular detection of nucleic acids by fluorescence signal amplification

An integrated PCR-free DNA sensor, which combines a sequence-specific receptor, an optical polymeric transducer, and an intrinsic fluorescence amplification mechanism, is reported. This sensor is based on the different conformations adopted by a cationic polythiophene when electrostatically bound to ss-DNA or ds-DNA, and on the efficient and fast energy transfer between the resulting fluorescent polythiophene/ds-DNA complex and neighboring fluorophores attached to ss-DNA probes. This molecular system allows the detection of only five molecules in 3 mL of an aqueous solution, or 3 zM, in 5 min. Moreover, this work demonstrates, for the first time, the direct detection of single nucleotide polymorphisms (SNPs) from clinical samples in only a few minutes, without the need for nucleic acid amplification.     

Infrared multiple photon dissociation spectroscopy of trapped ions

This tutorial review presents the technique of infrared multiple-photon dissociation (IRMPD) spectroscopy of mass-selected trapped ions. This requires coupling of a tunable infrared laser with mass spectrometry instrumentation. IRMPD spectroscopy has recently blossomed due to the emergence of widely tunable free electron lasers, as well as on-going developments of benchtop lasers. The merits of different trapping approaches in mass spectrometry are discussed in the light of photodissociation experiments. This tutorial discusses current capabilities, as well as limitations of the technique.     

Highly sensitive microRNA quantification with zero background signal from silver nanoparticles

MicroRNAs are a class of noncoding RNAs, which play vital roles in numerous cellular processes. Recent studies have confirmed their significance in the theranostics of various diseases. We herein fabricate an electrochemical approach for microRNA quantification. DNA/microRNA/DNA hybridization and electrochemical signals from silver nanoparticles (AgNPs) are employed in this work. DNA1 immobilized on a gold electrode interacts with target microRNA, along with amino group labeled DNA2, to form the sandwich hybrid. The adjacent DNA1 and DNA2 are then ligated, which can keep DNA2 on the electrode surface during the denaturation. Amino group modified at the 5′ end of DNA2 captures AgNPs on the electrode surface, which provide sharp stripping peaks for microRNA quantification. This electrochemical approach offers a simple and sensitive platform for the detection of microRNA, which shows great utility in biomedical research and clinical diagnosis.     

Pulsed multi-wavelength excitation using fiber-in-capillary light emitting diode induced fluorescence detection in capillary electrophoresis

A novel instrument was developed using a multi-wavelength pulsed LED array with in-column optic-fiber induced fluorescence detection by capillary electrophoresis. The light from 2 different wavelength LEDs (450 nm and 480 nm) was pulsed for short intervals at high intensity. The beam from each LED was collimated and reshaped with the gradient index (GRIN) lens group to achieve a highly effective coupling between LED light source and an optical fiber. The optical fiber was placed inside the capillary for in-capillary LED-induced fluorescence detection. The advantages of this system were validated by the simultaneous determination of vitamin B2 and fluorescein. Detection limits for vitamin B2 and fluorescein were estimated to be 5 nM and 0.29 nM (S/N = 3), respectively. The relative standard deviations (RSDs, n = 6) of the both compounds for migration time and peak area were better than 0.83%, 2.20% and 1.21%, 2.75%, respectively. The method was applied to the determination of vitamin B2 in commercial tablets and fluorescein in fluorescein sodium injection and the recoveries obtained were in the range of 96.6-102.0% and 99.9-102.8%, respectively. It was also applied to human serum, where the recoveries were found to be in the range of 94.4-97.0% and 92.6-96.4%, respectively. The system has been successfully applied in separation and determination of the both biological samples with acceptable analytical performance.     

Improving signal to background ratio for on-the-fly fluorescence lifetime detection in capillary electrophoresis

On-the-fly fluorescence lifetime detection (OFLD) in capillary electrophoresis (CE) was previously demonstrated using a commercial multiharmonic Fourier transform (MHF) spectrofluorometer interfaced to a commercial CE system. This paper discusses optimization of the interface design for minimization of background fluorescence and scattered light, thereby maximizing the signal-to-background ratio (S/B) of the dynamic measurement. Strategies included using various combinations of optical filters including a holographic filter and longpass or bandpass filters, tilting the capillary relative to the incident laser beam, employing a confocal design and adding an iris to remove out-of-focus light, using a microscope objective in the emission beam to increase the collection efficiency, and using square instead of ciruclar capillary columns. Significant improvements in S/B for on-column, on-the-fly detection of fluorescein in CE were achieved with most modifications.