Sample depletion of the matrix-assisted laser desorption process monitored using radionuclide detection

To investigate analyte consumption during the laser desorption process, matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is combined with radionuclide detection. Radionuclide detection provides highly sensitive and quantitative information on the amount of radiolabeled analytes in a MALDI MS sample spot. 14C-Labeled cytochrome c is deposited with 2,5-dihydroxybenzoic acid in 10-nL volume spots. By comparing radioactivity levels of the labeled cytochrome c both before and after spectral acquisition, the reduction in labeled analyte molecules on the target allows monitoring of the moles of desorbed sample. Through a depletion study on this sample, the amount of analyte consumed for MALDI time-of-flight spectral acquisition and the average number of molecules desorbed per laser ablation are determined. When [14C]-cytochrome c is no longer detected by MALDI MS, approximately 70% of the original analyte remains in the sample spots. Redissolving the spots produced further desorption, indicating that the analyte before dissolution was in a physical environment that did not facilitate the desorption process. As a technique with a response that does not depend on the environment of the analyte, radionuclide detection allows characterization of mass-limited sampling methods to better understand the MALDI process.     

Dynamics within site selectively templated and tagged xerogel sensor platforms

In a nitrobenzo-2-oxa-1,3-diazole (NBD) -based, 9-anthrol-responsive site selectively templated and tagged xerogel (SSTTX) sensor platform, there are two reporter molecule site types (responsive and non-responsive) that are responsible for the observed fluorescence signals. These NBD sites function independently. Site 1 alone binds the target analyte and yields an analyte-dependent signal. This signal arises from analyte binding decreasing the photo-induced electron transfer (PET) efficiency between a strategically placed amine residue and the excited NBD reporter molecule within the template site. Site 2 does not respond to analyte, it is not fully formed, and it manifests itself as a background signal. In an n-octyl residue-free SSTTX, the local microviscosity sensed by the site 1 NBD reporter molecules in the absence and presence of target analyte is ～260 cP and ～540 cP, respectively. These local microviscosity values are substantially greater in comparison to free NBD dissolved in THF (η = 0.46 cP at 298 K, ? ～25 ps). As the SSTTX n-octyl content is increased, the local microviscosity sensed by the site 1 NBD reporter molecules in the absence and presence of target analyte is ～360 cP and ～760 cP, respectively. This behavior is consistent with the n-octyl chains crowding the cybotactic region surrounding the site 1 NBD reporter molecules. This n-octyl-induced site 1 "crowding" is also associated with improved analyte binding to site 1 and better overall SSTTX analytical performance.     

Applicability of imaging time-of flight secondary ion MS to the characterization of solid-phase synthesized combinatorial libraries

We employ imaging time-of-flight secondary ion mass spectrometry to perform high-throughput analysis of solid-phase synthesized combinatorial libraries by acquiring mass spectra from arrays of polymer resin particles. To generalize this procedure to various types of resins and their associated chemical linkers, it is necessary to understand the dynamics associated with the analyte molecules during chemical pretreatment steps. Using stearic acid as a model compound, we examine the influence of three classes of linkers-acid or base labile linkers, a thermally labile linker, and a photochemically cleavable linker- all of which are used to anchor one end of the analyte to the polymer resin. With data obtained using secondary ion mass spectrometry, scanning electron microscopy, and X-ray photoelectron spectroscopy, we conclude that an effective treatment of the resin needs to include cleaving the linker and extracting the unbound analyte to the resin surface. We also demonstrate that the hydrophilicity of the polymeric constituents of a resin particle affects the experiments by changing the location of the analyte molecules during resin treatment. With this information, it is possible to utilize imaging TOF-SIMS to assay a range of material supports with assurance that high-quality spectra can be acquired.     

Targeted analyte detection by standard addition improves detection limits in matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) has proven an effective tool for fast and accurate determination of many molecules. However, the detector sensitivity and chemical noise compromise the detection of many invaluable low-abundance molecules from biological and clinical samples. To challenge this limitation, we developed a targeted analyte detection (TAD) technique. In TAD, the target analyte is selectively elevated by spiking a known amount of that analyte into the sample, thereby raising its concentration above the noise level, where we take advantage of the improved sensitivity to detect the presence of the endogenous analyte in the sample. We assessed TAD on three peptides in simple and complex background solutions with various exogenous analyte concentrations in two MALDI matrices. TAD successfully improved the limit of detection (LOD) of target analytes when the target peptides were added to the sample in a concentration close to optimum concentration. The optimum exogenous concentration was estimated through a quantitative method to be approximately equal to the original LOD for each target. Also, we showed that TAD could achieve LOD improvements on an average of 3-fold in a simple and 2-fold in a complex sample. TAD provides a straightforward assay to improve the LOD of generic target analytes without the need for costly hardware modifications.     

From mono- to polytopic interactions via hydrogen bonds — Capacitive sensor studies

Supra molecular materials can be generated via hydrogen bonding between monomers. This leads either to “monotopic” molecules as well as self-assemblies of a restricted number of complementary components as “di-” or “polytopic” molecules with more than one functional unit. The latter structures grow spontaneously by self organization and in principle consist of repeatedly branched chain molecules analogous to dendrimers. When disturbing these aggregates by analyte molecules (e.g. volatile organic compounds), the supra molecular structures break down leading to a pronounced change in dielectric behavior that is detectable with inter digital capacitors. “Monotopic”-materials show linear behavior with increasing analyte concentration whereas “polytopic” molecules lead to properties resembling a threshold sensor.     

Using volatile solvents for ion formation in liquid molecular beam expansion mass spectrometry

Cocrystallization between analyte and matrix is required by matrix-assisted laser desorption/ionization and can represent a significant limitation of the technique. A molecular beam expansion, mass spectrometric method has been developed to explore the possibility of using pure solvents as matrix to avoid cocrystallization. Two kinds of solvent, liquid CS2 and liquid or supercritical CO2, have been studied with 266-nm UV laser irradiation. We successfully ionized a number of compounds, including caffeine, guanine, cholesterol, and mixed fullerenes. Under some conditions, the mass spectra reflect parent radical cations formed by photoionization. Under other conditions, protonated, sodiated (and with CS2 even sulfated) ions are seen reflecting a nonunimolecular ionization process. When UV-transparent CO2 is used as a solvent, only analyte molecules with a UV chromophore are detected. However, with UV-absorbing CS2, we demonstrate ionization of molecules lacking a UV chromophore. This work provides strong evidence that one can form solvent clusters containing analyte, that laser photoionization of the solvent precedes ionization of the analyte, and that solvent evaporation along with the indirect ionization leads to reduced parent ion fragmentation. The exploration of this now demonstrated concept with other solvents would appear fruitful for future work.     

Detection limits for nanoscale biosensors

We examine through analytical calculations and finite element simulations how the detection efficiency of disk and wire-like biosensors in unmixed fluids varies with size from the micrometer to nanometer scales. Specifically, we determine the total flux of DNA-like analyte molecules on a sensor as a function of time and flow rate for a sensor incorporated into a microfluidic system. In all cases, sensor size and shape profoundly affect the total analyte flux. The calculations reveal that reported femtomolar detection limits for biomolecular assays are very likely an analyte transport limitation, not a signal transduction limitation. We conclude that without directed transport of biomolecules, individual nanoscale sensors will be limited to picomolar-order sensitivity for practical time scales.     

Digital concentration readout of single enzyme molecules using femtoliter arrays and Poisson statistics

Methods for accurately quantifying the concentration of a particular analyte in solution are all based on ensemble responses in which many analyte molecules give rise to the measured signal. In this paper, single molecules of beta-galactosidase were monitored using a 1 mm diameter fiber optic bundle with 2.4 x 10(5) individually sealed, femtoliter microwell reactors. By observation of the buildup of fluorescent products from single enzyme molecule catalysis over the array of reaction vessels and by application of a Poisson statistical analysis, a digital concentration readout was obtained. This approach should prove useful for single molecule enzymology and ultrasensitive bioassays. More generally, the ability to determine concentration by counting individual molecules offers a new approach to analysis of dilute solutions.     

Metal salts for molecular ion yield enhancement in organic secondary ion mass spectrometry: a critical assessment

In a search for molecular ion signal enhancement in organic SIMS, the efficiency of a series of organic and inorganic salts for molecular cationization has been tested using a panel of nonvolatile molecules with very different chemical characteristics (leucine enkephalin, Irganox 1010, tetraphenylnaphthalene, polystyrene). The compounds used for cationization include alkali bromide and group Ib metal salts (XBr with X = Li, Na, K; CF3CO2Ag; AgNO3; [CH3COCH=C(O-)CH3]2Cu; AuCl3). Alkali ions, very good for polar molecule cationization, prove to be of limited interest for nonpolar molecules such as polystyrene. Silver trifluoroacetate displays excellent results for all the considered molecules, except for leucine enkephalin (which might be due to the use of different solvents for the analyte and the salt). Instead, silver nitrate mixed with leucine enkephalin in an ethanol solution provides intense molecular signals. The influence of the respective concentrations of analyte and salt in solution, of the silver trifluoroacetate solution stability, and of the sample microstructure on the secondary ion intensities are also investigated. The results of other combinations of analyte and salts are reported. Finally, the use of salts is critically compared to other sample preparation procedures previously proposed for SIMS analysis of large organic molecules.     

A general method to perform a noncompetitive immunoassay for small molecules.

A new general method to perform a noncompetitive immunoassay for low-molecular-mass analytes (less than 6000 Da) is described and checked using cortisol as a model system. The method is based on the use of a "polydentate ligand" (cortisol-poly(L-lysine) conjugate) able to block the antibody sites unoccupied by the analyte, followed by the replacement of an antibody-bound analyte by an enzyme-labeled analyte (cortisol-horseradish peroxidase), and permits the direct measurement of the analyte bound sites. The observed signal shows a near-linear correlation with the analyte concentration. The characteristics of interactions between the analyte and polydentate ligand with the specific antibody were studied to perform a preliminary evaluation of the noncompetitive immunoassay for cortisol. The noncompetitive assay was compared with a competitive immunoassay obtained under the same conditions and using the same reagents. The results of the experiments showed a lower detection limit for the noncompetitive model (0.15 ng mL-1 rather than 0.72 ng mL-1), emphasizing that the model is successful. Moreover, as the polydentate ligand is prepared from the same hapten used for the immunogen synthesis, this type of noncompetitive immunoassay appears generally applicable to all small molecules for which antibodies have been obtained.     

MALDI-TOF mass spectrometry of insoluble giant polycyclic aromatic hydrocarbons by a new method of sample preparation

The insolubility of giant polycyclic aromatic hydrocarbons (PAHs) prevents their characterization by conventional analytical methods, which require a solubilization of the analyte. Laser desorption mass spectrometry may be used to analyze insoluble samples but is limited to relatively low molecular weights (approximately 2000), in the case of PAHs. To overcome this limitation, we applied MALDI-TOF mass spectrometry. Since MALDI sample preparation also requires solubility of analyte and matrix molecules, the sample preparation needed modification. The giant PAHs (>2000 Da) were investigated after using a new sample preparation, consisting of mechanically mixing analyte and matrix without any solubilization procedures. This solvent-free process allows insoluble compounds to be characterized. Furthermore, new organic molecules can be used as a matrix. Indeed, 7,7,8,8-tetracyanoquinodimethane, a new matrix with promising properties, has proven to be particularly suitable for the measurement of PAHs. Thanks to the successful characterization with MALDI-TOF mass spectrometry, the chemical design of giant PAHs, which was hindered until now for a lack of analytical methods, can now continue to develop.     

Liquid waveguide-based evanescent wave sensor that uses two light sources with different wavelengths

We demonstrate a prototypic optofluidic evanescent wave sensor made of poly(dimethylsiloxane) (PDMS) elastomer in which two light sources with different wavelengths are coupled into an optofluidic liquid-core/liquid-cladding (L(2)) waveguide. The exponentially decaying evanescent wave interacts with analyte molecules dissolved in the cladding fluids or products formed by in situ reactions at the core-cladding interface. The analyte molecules exhibit distinctly different light absorbance at the two wavelengths during the light-analyte interaction. Therefore, by using the normalized absorbance calculated from the intensity ratio of the two wavelengths instead of the absolute magnitude of either signal, unwanted effects from omnipresent external noise sources can be reduced. In addition, the differential absorption of the two beams by the analyte solutions can be used to enhance the resolution of sample analysis. The evanescent wave sensor based on a liquid waveguide can also be used for real-time monitoring of chemical reactions, because the core and cladding fluids in the L(2) waveguide are slightly miscible at the core-cladding interface due to the diffusional mixing.     

Compartmentalization of electrophoretically separated analytes in a multiphase microfluidic platform

Herein, we describe the monolithic integration of a multiphase microfluidic system to a microcapillary gel electrophoresis (μCGE) architecture for the complete isolation and storage of separated analyte bands. Within this platform, analyte molecules are separated using microchannel gel electrophoresis, and the eluted bands are stored in a sequence of approximately 40-600 encapsulating microdroplets. Importantly, employing such a system allows for total control of droplet size, shape, and composition. This approach is utilized to separate, optically detect, and encapsulate two fluorescent analytes from a composite sample mixture. Further to this, we subsequently investigate the potential of the system to be used as a concentration gradient generator through analysis of the segmented analyte bands and droplet composition.     

Real-time detection of organic compounds in liquid environments using polymer-coated thickness shear mode quartz resonators

The selection of sensitive coatings is a critical task in the design and implementation of chemical sensors using coated thickness shear mode quartz crystal resonators (QCRs) for detection in liquid environments. This design or selection is performed through a study of the sorption process in terms of the partition coefficients of the analytes in the coatings. The partition coefficient, which is controlled by the chemical and physical properties of the coating materials, determines the inherent selectivity and sensitivity toward analyte molecules. The selection of the coatings is logically determined by the interactions between coating and target analyte molecules, but can also be made through a systematic variation of the coating's properties. The determination of the partition coefficients is only accurate if all contributions to the total measured frequency shifts, deltafs, of the coated QCR can be established. While mass loading is often assumed to be the dominant factor used in determining partition coefficients, viscoelastic effects may also contribute to deltafs. Both the effect of viscoelastic properties and the effect of mass loading on the sensor responses are investigated by using a network analyzer and oscillator circuit and by characterizing the total mechanical impedance of the loaded sensor. Different types of coatings including rubbery and glassy polymers are investigated, and the targeted analytes include classes of polar compounds (methanol), nonpolar compounds (toluene, xylenes), and chlorinated hydrocarbons (trichloroethylene, tetrachloroethylene, etc). It is seen that changes in viscoelastic properties due to analyte sorption may be significant enough to place the sensor in the nongravimetric regime. However, for most applications involving the detection of relatively low concentrations of organic compounds and the use of acoustically thin films, changes in the complex shear modulus of the coatings contribute less than 5% of the total shift in the series resonant frequency, depending on the coating. In that case, the measured deltafs and, hence, the calculated approximate classification and selection of the coatings for operation in a complex solution of water/analyte molecules.     

Concentration gradient immunoassay. 2. Computational modeling for analysis and optimization

A novel microfluidic surface-based competition immunoassay, termed the concentration gradient immunoassay (described in detail in a companion paper (Nelson, K.; Foley, J.; Yager, P. Anal. Chem. 2007, 79, 3542-3548.) uses surface plasmon resonance (SPR) imaging to rapidly measure the concentration of small molecules. To conduct this assay, antibody and analyte are introduced into the two inlets of a T-sensor (Weigl, B. H.; Yager, P. Science 1999, 283, 346-347. Kamholz, A. E.; Weigl, B. H.; Finlayson, B. A.; Yager, P. Anal. Chem. 1999, 71, 5340-5347). Several millimeters downstream, antibody molecules with open binding sites can bind to a surface functionalized with immobilized antigen. This space- and time-dependent binding can be sensitively observed using SPR imaging. In this paper, we describe a complex three-dimensional finite element model developed to better understand the dynamic processes occurring with this assay. The model shows strong qualitative agreement with experimental results for small-molecule detection. The model confirms the experimental finding that the position within the microchannel at which the antibody binds to the immobilized analyte may be used to quantify the concentration of analyte in the sample. In addition, the model was used to explore the sensitivity of assay performance to parameters such as antibody and analyte concentrations, thereby giving insight into ways to optimize analysis speed and accuracy. Given the experimental verification of the computational results, this model serves as an efficient method to explore the influence of the flow rate, microchannel dimensions, and antibody concentration on the sensitivity of the assay.     

Fluorescence assay based on preconcentration by a self-ordered ring using berberine as a model analyte

A novel assay for trace amounts of fluorescent analytes is proposed based on the assembly of a self-ordered ring (SOR) through capillary flow in a sessile droplet on a glass slide support. After solvent evaporation of the sessile droplet containing a fluorescent analyte on a hydrophobic-treated glass slide, an outward capillary flow of the solvent from the interior of the droplet occurs. The resultant outward capillary flow then carries the analyte to the perimeter of the droplet spot where the analyte deposits and forms a fluorescent SOR. For the model analyte of berberine, SORs with outer diameter less than 1.2 mm and ring belt width less than 19 microm can be obtained depending on the droplet volume of the berberine solution. Data analysis for the digitally imaged SOR by using a CCD camera showed that the berberine molecules across the SOR belt section follow a Gaussian distribution, and the maximum fluorescent intensity (Imax) was found to be proportional to berberine content at the femtomole level. With the proposed technique, the content in tablets and the average excretion rates of berberine through human urine after oral administration could be satisfactorily monitored.     

Nanofiltration and sensing of picomolar chemical residues in aqueous solution using an optical porous resonator in a microelectrofluidic channel

For the first time the use of a porous microresonator placed in a microelectrofluidic system for integrated functions of nanofiltration and sensing of small biomolecules and chemical analytes in extremely dilute solution was proposed and investigated. As an example, aminoglycosides in drug residues in food and livestock products were considered as the trace chemical analyte. The filtration process of the charged analyte in aqueous solution driven by an applied electrical field and the accompanying optical whispering-gallery modes in the resonator are modeled. The dynamic process of adsorption and desorption of the analyte onto the porous matrix is studied. Deposition of the analyte inside the porous structure will alter the material refractive index of the resonator, and thus induce an optical resonance frequency shift. By measuring the optical frequency shift, the analyte concentration as well as the absorption/desorption process can be analyzed. Through an intensive numerical study, a correlation between the frequency shift and the analyte concentration and the applied electrical voltage gradient was obtained. This reveals a linear relationship between the resonance frequency shift and the analyte concentration. The applied electrical voltage substantially enhances the filtration capability and the magnitude of the optical frequency shift, pushing the porous resonator-based sensor to function at the extremely dilute picomolar concentration level for small bio/chemical molecules down to the sub-nanometer scale. Moreover, use of the second-order whispering-gallery mode is found to provide better sensitivity compared with the first-order mode.     

Laser desorption combined with hyperthermal surface ionization time-of-flight mass spectrometry

A setup combining laser desorption of nonvolatile molecules and their aerodynamic acceleration in a supersonic molecular beam followed by hyperthermal surface ionization in a reflectron time-of-flight mass spectrometer is described. While laser desorption performs the intact transfer of the analyte molecules into the gas phase, hyperthermal surface ionization opens up the possibility to efficiently ionize even larger molecules with a small and potentially controlled degree of fragmentation. Being an ionization technique, which is particularly effective for aromatic and heterocyclic compounds, the selectivity can further be increased by tuning the kinetic energy to which the molecules are accelerated in the supersonic beam. The results obtained for several polycyclic aromatic hydrocarbons and biochemical substances show that sufficient acceleration can be achieved even for molecules with a molecular weight above 5000 amu and that HSI preserves its advantageous features even for thermally labile large molecules such as insulin.     

Electrospray-assisted laser desorption/ionization mass spectrometry for continuously monitoring the states of ongoing chemical reactions in organic or aqueous solution under ambient conditions

Electrospray-assisted laser desorption/ionization (ELDI) combined with mass spectrometry allows chemical and biochemical compounds to be characterized directly from hydrophilic and hydrophobic organic solutions mixed with carbon powders under ambient conditions. Organic and inorganic compounds dissolved in polar or nonpolar solvent such as methanol, tetrahydrofuran, ethyl acetate, toluene, dichloromethane, or hexane can be detected using this ambient ionization technique without prior pretreatment. We have used this technique to monitor the progress in several ongoing reactions: the epoxidation of chalcone in ethanol, the chelation of ethylenediaminetetraacetic acid with copper and nickel ions in aqueous solution, the chelation of 1,10-phenanthroline with iron(II) in methanol, and the tryptic digestion of cytochrome c in aqueous solution. Liquid-ELDI analyses simply require irradiation of the surface of the sample solution with a pulsed ultraviolet laser; the laser energy is adsorbed by the carbon powder presuspended in the sample solution; the absorbed laser energy is then transferred to the surrounding solvent and to the analyte molecules in the solution, leading to their desorption; the desorbed gaseous analyte molecules are then postionized within an electrospray (ESI) plume to generate ESI-like analyte ions.     

Identification of the carboxylic acid functionality by using electrospray ionization and ion-molecule reactions in a modified linear quadrupole ion trap mass spectrometer

A mass spectrometric method has been developed for the identification of the carboxylic acid functional group in analytes evaporated and ionized by electrospray ionization (ESI). This method is based on gas-phase ion-molecule reactions of ammoniated ([M + NH4]+) and sodiated ([M + Na]+) analyte molecules with trimethyl borate (TMB) in a modified linear quadrupole ion trap mass spectrometer. The diagnostic reaction involves addition of the deprotonated analyte to TMB followed by the elimination of methanol. A variety of analytes with different func-tionalities were examined, and this reaction was only observed for molecules containing the carboxylic acid functionality. The selectivity of the reaction is attributed to the acidic hydrogen present in the carboxylic acid group, which provides the proton necessary for the elimination of methanol. The diagnostic products are easily identified based on the m/z value of the product ion, which is 72 Th (thomson) greater than the m/z value of the charged analyte, and also by the character-istic isotope pattern of boron. The applicability of this method for pharmaceutical analysis was demonstrated for three nonsteroidal anti-inflammatory drugs: ibuprofen, naproxen, and ketoprofen.