Small Molecule Analytes Research Articles

Continuous, Real-Time Monitoring of Cocaine in Undiluted Blood Serum via a Microfluidic, Electrochemical Aptamer-Based Sensor

The development of a biosensor system capable of continuous, real-time measurement of small-molecule analytes directly in complex, unprocessed aqueous samples has been a significant challenge, and successful implementation has been achieved for only a limited number of targets. Toward a general solution to this problem, we report here the Microfluidic Electrochemical Aptamer-based Sensor (MECAS) chip wherein we integrate target-specific DNA aptamers that fold, and thus generate an electrochemical signal, in response to the analyte with a microfluidic detection system. As a model, we demonstrate the continuous, real-time (approximately 1 min time resolution) detection of the small-molecule drug cocaine at near physiological, low micromolar concentrations directly in undiluted, otherwise unmodified blood serum. We believe our approach of integrating folding-based electrochemical sensors with miniaturized detection systems may lay the groundwork for the real-time, point-of-care detection of a wide variety of molecular targets.

Preparation of plasma samples for chromatographic analyses using functionalized ferromagnetic micro-particles manipulated in a high pressure liquid system

Objectives To study if functionalized ferromagnetic micro-particles used for extraction procedures prior to chromatographic analyses can be handled within an HPLC system. Method We used C18-functionalized ferromagnetic micro-particles, manipulated within a high-performance liquid chromatography system for extraction of plasma samples, with a novel extractor device enabling reversible immobilization of the particles within HPLC tubing. A pilot analytical system for the quantification of itraconazole in plasma – as a representative small molecule analyte – by ultraviolet detection was configured and tested. Results The system allowed linear and reproducible quantification of the representative target analyte. Conclusions Sample preparation of biological samples can be achieved by handling of functionalized ferromagnetic micro-particles which are handled within a high pressure liquid chromatography configuration; this offers interesting perspectives for the development of automated sample preparation devices.

Dried blood spots as a source of anti-malarial antibodies for epidemiological studies

BackgroundBlood spots collected onto filter paper are an established and convenient source of antibodies for serological diagnosis and epidemiological surveys. Although recommendations for the storage and analysis of small molecule analytes in blood spots exist, there are no published systematic studies of the stability of antibodies under different storage conditions.MethodsBlood spots, on filter paper or glass fibre mats and containing malaria-endemic plasma, were desiccated and stored at various temperatures for different times. Eluates of these spots were assayed for antibodies against two Plasmodium falciparum antigens, MSP-119 and MSP2, and calculated titres used to fit an exponential (first order kinetic) decay model. The first order rate constants (k) for each spot storage temperature were used to fit an Arrhenius equation, in order to estimate the thermal and temporal stability of antibodies in dried blood spots. The utility of blood spots for serological assays was confirmed by comparing antibodies eluted from blood spots with the equivalent plasma values in a series of samples from North Eastern Tanzania and by using blood spot-derived antibodies to estimate malaria transmission intensity in this site and for two localities in Uganda.ResultsAntibodies in spots on filter paper and glass fibre paper had similar stabilities but blood was more easily absorbed onto filter papers than glass fibre, spots were more regular and spot size was more closely correlated with blood volume for filter paper spots. Desiccated spots could be stored at or below 4°C for extended periods, but were stable for only very limited periods at ambient temperature. When desiccated, recoveries of antibodies that are predominantly of IgG1 or IgG3 subclasses were similar. Recoveries of antibodies from paired samples of serum and of blood spots from Tanzania which had been suitably stored showed similar recoveries of antibodies, but spots which had been stored for extended periods at ambient humidity and temperature showed severe loss of recoveries. Estimates of malaria transmission intensity obtained from serum and from blood spots were similar, and values obtained using blood spots agreed well with entomologically determined values.ConclusionThis study has demonstrated the suitability of filter paper blood spots paper for collection of serum antibodies, and provided clear guidelines for the treatment and storage of filter papers which emphasize the importance of desiccation and minimisation of time spent at ambient temperatures. A recommended protocol for collecting, storing and assaying blood spots is provided.

Protection of Sensors for Biological Applications by Photoinitiated Chemical Vapor Deposition of Hydrogel Thin Films

We report photoinitiated chemical vapor deposition (piCVD), a gentle synthetic method for the preparation of ultrathin films (approximately 100 nm) of the hydrogel poly(hydroxyethyl methacrylate) (pHEMA). piCVD occurs near room temperature and requires only mild vacuum conditions. The deposited films swell rapidly and reversibly in buffer solution, and the swelling properties can be controlled via the deposition conditions. Analysis of the swelling data indicates that the mesh size of the hydrogel creates a selectively permeable coating. The mesh is large enough to allow small molecule analytes to permeate the film but small enough to prevent the transport of large biomolecules such as proteins. X-ray photoelectron spectroscopy (XPS) shows that the films decrease nonspecific adhesion of the protein albumin by nearly 8-fold over bare silicon. A dry process, piCVD is suitable for coating particles with diameters as small as 5 microm. The absence of solvents and plasmas in piCVD allows films to be directly synthesized on optode sensors without degradation of sensitivity or response time.

Sample preparation for liquid chromatography-tandem mass spectrometry using functionalized ferromagnetic micro-particles

Objectives To study the applicability of functionalized ferromagnetic micro-particles in the sample preparation for quantification of small molecules by LC-MS/MS. Design and methods A representative analyte (itraconazole) was extracted from plasma samples using small quantities of C18-modified ferromagnetic micro-particles. Results 125 μg of functionalized micro-particles was sufficient to extract the target analyte from 10 μL of plasma with a recovery rate of approximately 100%; linearity ( r > 0.99) and reproducibility (inter-assay CV ≤ 7%) of quantitative results was found. Conclusions C18-functionalized ferromagnetic particles can be used to efficiently extract small molecule analytes from complex biological matrices for quantitative analyses using LC-MS/MS.

Trace Level Detection of Compounds Related to the Chemical Weapons Convention by 1H-Detected 13C NMR Spectroscopy Executed with a Sensitivity-Enhanced, Cryogenic Probehead

Two-dimensional 1H-13C HSQC (heteronuclear single quantum correlation) and fast-HMQC (heteronuclear multiple quantum correlation) pulse sequences were implemented using a sensitivity-enhanced, cryogenic probehead for detecting compounds relevant to the Chemical Weapons Convention present in complex mixtures. The resulting methods demonstrated exceptional sensitivity for detecting the analytes at trace level concentrations. 1H-13C correlations of target analytes at < or = 25 microg/mL were easily detected in a sample where the 1H solvent signal was approximately 58,000-fold more intense than the analyte 1H signals. The problem of overlapping signals typically observed in conventional 1H spectroscopy was essentially eliminated, while 1H and 13C chemical shift information could be derived quickly and simultaneously from the resulting spectra. The fast-HMQC pulse sequences generated magnitude mode spectra suitable for detailed analysis in approximately 4.5 h and can be used in experiments to efficiently screen a large number of samples. The HSQC pulse sequences, on the other hand, required roughly twice the data acquisition time to produce suitable spectra. These spectra, however, were phase-sensitive, contained considerably more resolution in both dimensions, and proved to be superior for detecting analyte 1H-13C correlations. Furthermore, a HSQC spectrum collected with a multiplicity-edited pulse sequence provided additional structural information valuable for identifying target analytes. The HSQC pulse sequences are ideal for collecting high-quality data sets with overnight acquisitions and logically follow the use of fast-HMQC pulse sequences to rapidly screen samples for potential target analytes. Use of the pulse sequences considerably improves the performance of NMR spectroscopy as a complimentary technique for the screening, identification, and validation of chemical warfare agents and other small-molecule analytes present in complex mixtures and environmental samples.

Conditioning saliva for use in a microfluidic biosensor

This report details an approach to saliva conditioning for compatibility of raw patient samples with microfluidic immunoassay components, principally biosensor surfaces susceptible to fouling. Stimulated whole human saliva spiked with a small molecule analyte (phenytoin, 252 Da) was first depleted of cells, debris and high molecular weight glycoproteins (mucins) using membrane filtration. This process significantly reduced but did not eliminate fouling of biosensor surfaces exposed to the sample. An H-filter, which separates solutes from mixed samples based on their diffusion in laminar flow, was used to extract the analyte from the remaining large molecular weight species in the filtered saliva sample. Patient samples treated in this way retained 23% of the analyte with 97% and 92% reduction in glycoproteins and proteins, respectively, and resulted in 3.6 times less surface fouling than either untreated or filtered saliva alone. These sample conditioning steps will enable the use of fouling-sensitive detection techniques in future studies using clinical saliva samples.

Surface plasmon resonance for probing quadruplex folding and interactions with proteins and small molecules

Surface plasmon resonance is a technique for detecting binding events at the surface of a thin metal film. Through the commercial availability of instrumentation and sensor chips, the technique has found widespread application for determining the affinity and kinetics of macromolecular interactions. A variety of quadruplex forming oligonucleotides have been immobilized to sensor chips to permit analysis of their binding interactions with both small molecule and protein analytes. The fold of the quadruplex must be maintained through an appropriate choice of buffer, and care must be taken to ensure that data interpretation is not hampered by non-specific binding and adsorption of the analyte to the sensor surface and instrument. Affinity constants determined by surface plasmon resonance for interactions with quadruplexes correlate meaningfully with other methods, such as UV–visible and fluorescence titrations, enzyme linked immunosorbent assay, thermal melting studies and telomerase inhibition. Kinetic measurements of the association and dissociation of duplexes of quadruplex forming oligonucleotides and their complementary strands have enabled calculation of the folding and unfolding rates of the quadruplex itself, and determination of its stability as a function of buffer composition.

Thermoset polyester as an alternative material for microchip electrophoresis/electrochemistry

Microchip CE coupled with electrochemical detection (MCE-EC) is a good method for the direct detection of many small molecule analytes because the technique is sensitive and readily miniaturized. Polymer materials are being increasingly used with MCE due to their affordability and ease of fabrication. While PDMS has become arguably the most widely used material in MCE-EC due to the simplicity of microelectrode incorporation, it suffers from a lack of separation efficiency, lower surface stability, and a tendency for analyte sorption. Other polymers, such as poly(methylmethacrylate) (PMMA) and poly(carbonate) (PC), have higher separation efficiencies but require more difficult fabrication techniques for electrode incorporation. In this report, thermoset polyester (TPE) was characterized as an alternative material for MCE-EC. TPE microchips were characterized in their native and plasma oxidized forms and after coating with polyelectrolyte multilayers (PEMs). TPE provides higher separation efficiencies when compared to PDMS microchips, while still using simple fabrication protocols. In this work, separation efficiencies as high as 295,000 N/m were seen when using TPE MCE-EC devices. Furthermore, the EOF was higher and more consistent as a function of pH for both native and plasma-treated TPE than PDMS. Finally, TPE is amenable to modification using simple PEM coatings as another way to control surface chemistry and surface charge.

SPR Imaging‐Based Salivary Diagnostics System for the Detection of Small Molecule Analytes

Saliva is an underused fluid with considerable promise for biomedical testing. Its potential is particularly great for monitoring small-molecule analytes since these are often present in saliva at concentrations that correlate well with their free levels in blood. We describe the development of a prototype diagnostic device for the rapid detection of the antiepileptic drug (AED) phenytoin in saliva. The multicomponent system includes a hand-portable surface plasmon resonance (SPR) imaging instrument and a disposable microfluidic assay card.

Electron Donor Solvent Effects Provide Biosensing with Quantum Dots

A palmitate biosensor that uses the emission intensity of a semiconducting nanoparticle to report palmitate concentration is presented. This method uses electron transfer to quench the emission from a ZnS-coated CdSe nanoparticle. The fatty acid binding pocket of intestinal fatty acid binding protein is used to modulate the electron transfer properties of [Ru(L)(NH3)4](PF6)2 (L = 5-maleimido-1,10-phenanthroline) that is covalently attached within this pocket. Once the metal-complex-modified protein is attached to ZnS-coated CdSe nanoparticles, palmitate addition excludes water from around the metal complex and increases the electron transfer from the metal complex to the valence band hole of the nanoparticle excited state. A 1.6-fold change in emission intensity is observed upon adding a saturated amount (500 nM) of sodium palmitate. The dissociation constant was calculated as 5 nM with a 1 nM lower limit of detection. Since palmitate does not alter the global conformation of intestinal fatty acid binding protein, palmitate-mediated changes in pocket solvation are suggested. This represents a new method in biosensor construction with semiconducting nanoparticles. Including previous conformation-dependent biosensors, there are thousands of potential analytes that can be detected with these strategies. Such biosensors will provide fluorescence contrast imaging reagents for small molecule analytes.

Quantification and Identification of Components in Solution Mixtures from 1D Proton NMR Spectra Using Singular Value Decomposition

One-dimensional proton NMR spectra of complex solutions provide rich molecular information, but limited chemical shift dispersion creates peak overlap that often leads to difficulty in peak identification and analyte quantification. Modern high-field NMR spectrometers provide high digital resolution with improved peak dispersion. We took advantage of these spectral qualities and developed a quantification method based on linear least-squares fitting using singular value decomposition (SVD). The linear least-squares fitting of a mixture spectrum was performed on the basis of reference spectra from individual small-molecule analytes. Each spectrum contained an internal quantitative reference (e.g., DSS-d6 or other suitable small molecules) by which the intensity of the spectrum was scaled. Normalization of the spectrum facilitated quantification based on peak intensity using linear least-squares fitting analysis. This methodology provided quantification of individual analytes as well as chemical identification. The analysis of small-molecule analytes over a wide concentration range indicated the accuracy and reproducibility of the SVD-based quantification. To account for the contribution from residual protein, lipid or polysaccharide in solution, a reference spectrum showing the macromolecules or aggregates was obtained using a diffusion-edited 1D proton NMR analysis. We demonstrated this approach with a mixture of small-molecule analytes in the presence of macromolecules (e.g., protein). The results suggested that this approach should be applicable to the quantification and identification of small-molecule analytes in complex biological samples.

Self-referenced assay method for photonic crystal biosensors: Application to small molecule analytes

Using an image-based method for label-free detection of biochemical binding density distribution on the surface of a photonic crystal biosensor embedded within standard format multiwell microplates, a new method for automatic referencing of assay errors due to variability in the bulk refractive index of the analyte test sample is demonstrated. The new method involves application of the immobilized ligand upon the biosensor surface as a small spot, so that uncoated regions of a microplate well may serve as an accurate reference for the “active” regions containing the ligand. Due to the high spatial resolution of the image-based detection approach, each well in the microplate may be represented by hundreds of independent measurements of biochemical binding density, as measured by the shift in reflected wavelength from the photonic crystal biosensor. A linear plot of detected analyte density as a function of immobilized ligand density is constructed, in which the slope represents the ligand/analyte affinity and the intercept represents common-mode error effects. Streptavidin–biotin is used as a ligand–analyte model system to demonstrate the ability of this method to separate intentionally introduced bulk refractive index errors from the detection of a small molecule analyte. This referencing approach will be important in the context of small molecule drug compound library screening, where drug compounds within a large library are often suspended in solutions of inconsistent bulk refractive index.

Combining restricted access material (RAM) and turbulent flow for the rapid on-line extraction of the cyclooxygenase-2 inhibitor rofecoxib in plasma samples

Restricted access material (RAM) has been used in the packing of a solid-phase extraction (SPE) column for on-line extractions under turbulent flow conditions. The bio-compatible RAM material works by the principle of size exclusion in addition to conventional reversed-phase chromatography, thereby allowing the extraction and preconcentration of small analyte molecules from biological samples such as plasma. Using small column dimensions (0.76 mm × 50 mm) and a consequently high linear velocity, turbulent flow was achieved during online sample extractions. The improved mass-transfer rate characteristic of turbulent flow allows fast sample cleanup without decreased extraction efficiency. The novel use of the RAM column, connected upstream to a C 18 monolithic column, allowed the direct injection, extraction, separation, and MS/MS detection of plasma samples spiked with rofecoxib in a span of 5 min. Calibration curves obtained using this RAM turbulent flow coupled column method showed good linearity ( R 2 > 0.99) and reproducibility (%RSD ≤ 7%). The lower limit of quantitation of rofecoxib in plasma samples was found to be 40 ng/ml. The extraction method showed good recovery of rofecoxib from a plasma matrix with minimal signal loss and robustness after more than 200 plasma injections.

Fluorescence Quenching Mechanism of a Polyphenylene Polyelectrolyte with Other Macromolecules: Cytochrome c and Dendrimers

This study investigates the fluorescence quenching of a polyphenyl based polyelectrolyte by positively charged macromolecules (proteins and dendrimers). This work shows that the fluorescence quenching of the dendrimer materials does not involve energy transfer or electron transfer but is correlated to the overall charge on the dendrimer and its size. The quenching is hypothesized to result from conformational changes that occur upon binding the polyelectrolyte to the protein or dendrimer. This mechanism is qualitatively different from that invoked for small-molecule analytes.

Using electrospray ionization-mass spectrometry/tandem mass spectrometry and small molecules to study guanidinium–anion interactions

A previous study highlighting the interaction between guanidinium- and phosphonate-functionalized molecules and the development of a screening protocol for noncovalent interactions using ESI-MS and MS/MS methodologies is extended here to incorporate sulfonate- and carboxylate-functionalized binding partners for guanidinium. Multiple high order homomeric and heteromeric adduct ions are observed in the mass spectra when mixtures of complementary analytes are ionized. Comparison of relative binding and ionization efficiencies are made using the solution-phase competition methods and gas-phase collision threshold dissociation ( E 1/2) measurements. Transmission factors are determined to compare the effect of structural variation of the analytes on their relative ionization efficiencies. Results indicate that while phosphonate- and sulfonate-functionalized analytes form more and higher order adduct ion complexes with guanidinium-containing molecules (represented here by free and modified arginines) as a result of the ESI process, when solvent is removed and collisional dissociation is employed, the trend is reversed, and the carboxylate group yields a stronger interaction with guanidinium, relative to the other oxoanions. Ionization differences reflected in the mass spectra are attributed to pH effects present in the condensed phase, whereas differences in stability measured in the gas-phase are attributed to the gas-phase acidities of the oxoanions and their geometric complementarity when forming noncovalent interactions with guanidinium. This work highlights the interaction of guanidinium with oxoanion binding partners using various ESI-MS and MS/MS methods, but also addresses explicitly the advantages and disadvantages of using small molecule analytes for routine analysis of noncovalent interactions.

Luminescent Quantum Dot-Adaptor Protein-Antibody Conjugates for Use in Fluoroimmunoassays

A method for the preparation and characterization of bioinorganic conjugates made with highly luminescent semiconductor CdSe–ZnS core-shell quantum dots (QDs) and antibodies for use in fluoroimmunoassays is presented. The conjugation strategy employs two routes: 1. Use of an engineered molecular adaptor protein, attached to the QDs via electrostatic/hydrophobic self-assembly, to link the inorganic fluorophore with antibodies, and 2. use of avidin, also electrostatically self-assembled onto the nanocrystal surface, which allows QD conjugation to biotinylated antibodies via avidin–biotin binding scheme. With this approach, the average number of antibodies conjugated to a single QD can be varied. In addition, we have developed a simple purification strategy based on mixed composition conjugates of the molecular adaptor and a second “inert” two-domain fusion protein that allows the use of affinity chromatography. QD/adaptor-antibody conjugates were successfully employed in fluoroimmunoassays for the detection of small molecule analytes, 2,4,6-trinitrobenzene (TNB) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). We also demonstrate the use of QD/avidin-antibody conjugates for fluoroimmunoassays using a model protein system.

The use of high-flow high performance liquid chromatography coupled with positive and negative ion electrospray tandem mass spectrometry for quantitative bioanalysis via direct injection of the plasma/serum samples.

Two bioanalytical methods have been developed and validated utilizing high flow high performance liquid chromatography (HPLC) for on-line purification of plasma and serum samples and electrospray tandem mass spectrometry for detection and quantitation. Each plasma or serum sample, after mixing with an aqueous solution of the internal standard, was injected into a small diameter (1 x 50 mm) column packed with large particles of OASIS (30 microns), with a 100% aqueous mobile phase at a high flow rate (3-4 mL/min). The combination of the high linear speed (6-8 cm/s) of the aqueous mobile phase and the large particle size resulted in the rapid passage of the proteins and other large biomolecules through the column while the small-molecule analytes were retained on the column. During this purification period, the HPLC effluent was directed to waste. After the purification step, the HPLC mobile phase was rapidly changed from 100% aqueous to < or = 100% organic, the flow was reduced to 0.5-0.8 mL/min, and the column effluent was directed towards the mass spectrometer. The small molecule analytes were eluted during this period. In the method developed and validated for the quantitative determination of compound I in rat plasma (method A), the same OASIS column (1 x 50 mm, 30 microns) served as the purification and analytical (elution) column. In the method developed for the simultaneous determination of pravastatin and its positional isomer biotransformation product (SQ-31906) in human serum (method B), the purification column was connected to a conventional C18 analytical column (3.9 x 50 mm, 5 microns) to achieve the required chromatographic separation between the two isomers. For method A, where 50 microL of rat plasma mixed 1:1 with water containing the internal standard was injected, the standard curve range was 1 to 1,000 ng/mL. For method B, where 200 microL of a human serum sample mixed 4:1 with water containing the internal standard was injected, the standard curve range was 0.5 to 100 ng/mL. The total analysis time for each method was < or = 5 min per sample. The accuracy, inter-day precision and intra-day precision were within 10% for both methods.