Pollen discrimination and classification by Fourier transform infrared (FT-IR) microspectroscopy and machine learning

The discrimination and classification of allergy-relevant pollen was studied for the first time by mid-infrared Fourier transform infrared (FT-IR) microspectroscopy together with unsupervised and supervised multivariate statistical methods. Pollen samples of 11 different taxa were collected, whose outdoor air concentration during the flowering time is typically measured by aerobiological monitoring networks. Unsupervised hierarchical cluster analysis provided valuable information about the reproducibility of FT-IR spectra of the same taxon acquired either from one pollen grain in a 25 × 25 μm² area or from a group of grains inside a 100 × 100 μm² area. As regards the supervised learning method, best results were achieved using a K nearest neighbors classifier and the leave-one-out cross-validation procedure on the dataset composed of single pollen grain spectra (overall accuracy 84%). FT-IR microspectroscopy is therefore a reliable method for discrimination and classification of allergenic pollen. The limits of its practical application to the monitoring performed in the aerobiological stations were also discussed. Figure Traditional and innovative methods for the identification of airborne pollen grains     

Analysis of type I and IV collagens by FT-IR spectroscopy and imaging for a molecular investigation of skeletal muscle connective tissue

Many muscular diseases result from abnormal organization of connective tissue and/or collagen network formation. Only a few molecular imaging techniques are able to analyze this collagen network by differentiating collagen types. In this study, FT-IR spectroscopy was used to analyze type I and IV collagens, the most important compounds of which are perimysium and endomysium, respectively. Secondary structure of collagen types was determined by curve-fitting the 1,700–1,480 cm⁻¹ spectral interval. Type I collagen could be differentiated from type IV by its higher amounts of triple helix and α-helix, but lower amounts of β-sheets (P < 0.01). FT-IR imaging was then used to determine structural features of perimysium and endomysium collagen network in bovine Flexor carpi radialis muscle. Secondary structure of proteins contained in perimysium and endomysium was found to be very close to type I and IV collagens, respectively. FT-IR spectroscopy and imaging are thus analytical tools that might be used for investigating biodistribution and assembly of collagen types in connective tissues. Figure Visible (left) and full spectral FT-IR (right) images of skeletal muscle tissue section (16 μm) exhibiting a vertical arrangement of fibers. + and × in FT-IR image show selected positions to obtain FT-IR spectra of perimysium and endomysium, respectively     

Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine

A multianalyte lateral-flow immunochromatographic technique using colloidal gold-labeled polyclonal antibodies was developed for the rapid simultaneous detection of clenbuterol and ractopamine. The assay procedure could be accomplished within 5 min, and the results of this qualitative one-step assay were evaluated visually according to whether test lines appeared or not. When applied to the swine urines, the detection limit and the half maximal inhibitory concentration (IC₅₀) of the test strip under an optical density scanner were calculated to be 0.1 ± 0.01 ng mL⁻¹ and 0.1 ± 0.01 ng mL⁻¹, 0.56 ± 0.08 ng mL⁻¹, and 0.71 ± 0.06 ng mL⁻¹, respectively, the cut-off levels with the naked eye of 1 ng mL⁻¹ and 1 ng mL⁻¹ for clenbuterol and ractopamine were observed. Parallel analysis of swine urine samples with clenbuterol and ractopamine showed comparable results obtained from the multianalyte lateral-flow test strip and GC-MS. Therefore, the described multianalyte lateral-flow test strip can be used as a reliable, rapid, and cost-effective on-site screening technique for the simultaneous determination of clenbuterol and ractopamine residues in swine urine.      

Enhancing the analytical performance of immunoassays that employ metal-enhanced fluorescence

In this work, we used a model assay system (polyclonal human IgG–goat antihuman IgG) to elucidate some of the key factors that influence the analytical performance of bioassays that employ metal-enhanced fluorescence (MEF) using silver nanoparticles (NPs). Cy5 dye was used as the fluorescent label, and results were compared with a standard assay performed in the absence of NPs. Two sizes of silver NPs were prepared with respective diameters of 60 ± 10 and 149 ± 16 nm. The absorption spectra of the NPs in solution were fitted accurately using Mie theory, and the dipole resonance of the 149-nm NPs in solution was found to match well with the absorption spectrum of Cy5. Such spectral matching is a key factor in optimizing MEF. NPs were deposited uniformly and reproducibly on polyelectrolyte-coated polystyrene substrates. Compared to the standard assay performed without the aid of NPs, significant improvements in sensitivity and in limit of detection (LOD) were obtained for the assay with the 149-nm NPs. An important observation was that the relative enhancement of fluorescence increased as the concentration of antigen increased. The metal-assisted assay data were analyzed using standard statistical methods and yielded a LOD of 0.086 ng/mL for the spectrally matched NPs compared to a value of 5.67 ng/mL obtained for the same assay in the absence of NPs. This improvement of ∼66× in LOD demonstrates the potential of metal-enhanced fluorescence for improving the analytical performance of bioassays when care is taken to optimize the key determining parameters.      

Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay

Two kinds of Ca²⁺-regulated photoprotein obelin with altered color of bioluminescence were obtained by active-center amino acid substitution. The mutant W92F-H22E emits violet light (λ_max = 390 nm) and the mutant Y139F emits greenish light (λ _max = 498 nm), with small spectral overlap, both display high activity and stability and thus may be used as reporters. For demonstration, the mutants were applied in dual-color simultaneous immunoassay of two gonadotropic hormones—follicle-stimulating hormone and luteinizing hormone. Bioluminescence of the reporters was simultaneously triggered by single injection of Ca²⁺ solution, divided using band-pass optical filters and measured with a two-channel photometer. The sensitivity of simultaneous bioluminescence assay was close to that of a separate radioimmunoassay. Figure Two kinds of Ca²⁺-regulated photoprotein obelin with altered color of bioluminescence were obtained and applied in dual-color simultaneous immunoassay of two gonadotropic hormones.     

Determination of vancomycin in human plasma using high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the quantification of vancomycin in human plasma was developed and validated. The method includes an extraction of vancomycin by deproteinization with acetonitrile. The analyses were carried out at 258 nm as the emission wavelength while exciting at 225 nm on a reversed-phase column (30 cm × 4 mm i.d. × 10 μm Waters Associates μBondapak C18) using a mobile phase composed of methanol and phosphate buffer at pH 6.3. Vancomycin was quantitatively recovered from human plasma samples (>96%) with high values of precision. The separation was completed within 27 min. The calibration curve was linear over the range from 5 to 1,000 ng/mL with the detection and quantification limits of 2 ng/mL and 5 ng/mL, respectively. This method is suitable for the routine assay of plasma samples. Figure The effect of the deproteinization solvent on the signal of the interference peak at retention time of 15.0 min. The peak which interferes with the peaks of Erythromycin and Vancomycin has been disappeared by using 2 mL acetonitrile as the deproteinization solvent.     

High-throughput evaluation of quiescent hematopoietic progenitor cells using a micro-multiwell plate

Conventional assays for hematopoietic progenitor cells (HPCs) require long-term culturing, a labor-intensive procedure, and technique proficiency. We aimed to develop a high-throughput method to determine frequency of quiescent primitive HPCs by a combination of the micro-multiwell plate and 5-fluorouracil (5-FU) treatment. The micro-multiwell plate was made of a silicone sheet with a 6 × 6 array of 1-mm diameter holes and a glass substrate. To enrich primitive HPCs in a CD34 population, CD34 cells and stromal cells were applied to micro-multiwells and cultured in the presence of 5-FU for 2 days. The quiescent primitive HPCs that survived after 5-FU treatment were then expanded with cytokines in the absence of 5-FU for a further 10 days. After culturing, cells were immunostained and the number of primitive HPCs in inoculated CD34 cells was estimated from fluorescent intensity for each well under a stereoscopic fluorescent microscope. The frequencies of primitive HPCs correlated well with frequencies of cobblestone area-forming cells for two CD34 cell lots. Our method allows high-throughput screening for primitive HPCs in CD34 cells. Figure Representative image of a micro-multiwell plate fordetecting primitive hematopoietic stem cells     

Electrochemical deposition of silicate–cetyltrimethylammonium bromide nanocomposite film on glassy carbon electrode for sensing of methyl parathion

A novel electrochemical sensor for methyl parathion based on silicate– cetyltrimethylammonium bromide nanocomposite film has been fabricated by electro-assisted deposition onto glassy carbon electrode in one-step via an electrochemical modulation of pH at the electrode/solution interface to promote controlled gelification of tetraethylorthosilicate sol, and was characterized with scanning electron microscopy, X-ray diffraction, and electrochemical impedance spectroscopy. The electrochemical sensing of methyl parathion on the film-modified electrode was investigated applying cyclic voltammetry and square wave voltammetry. Compared to the unmodified electrode, the shapes of the redox peaks were improved and the peak currents significantly increased. Experimental parameters such as deposition time, pH value, and accumulation conditions have been optimized. A linear relationship between the peak current and methyl parathion concentration was obtained in the range from 1.0 × 10⁻⁷ to 1.0 × 10⁻⁴ mol L⁻¹ with a detection limit of 1.04 × 10 ⁻⁸ mol L⁻¹ (S/N = 3) after accumulation at 0 V for 120 s. The film electrode shows great promise for determination of methyl parathion in real samples.      

Forensic analysis of dyed textile fibers

A rapid, specific, and sensitive method has been developed using molecularly imprinted polymers (MIPs) as solid-phase extraction sorbents for extraction of trace tetracycline antibiotics (TCs) in foodstuffs. MIPs were prepared by precipitation polymerization using tetracycline as the template. Under the optimal condition, the imprinting factors for MIPs were 4.1 (oxytetracycline), 7.0 (tetracycline), 7.4 (chlortetracycline), 7.7 (doxycycline), respectively. Furthermore, the performance of MIPs as solid-phase extraction sorbents was evaluated and high extraction efficiency of molecularly imprinted solid-phase extraction (MISPE) procedure was demonstrated. Compared with commercial sorbents, MISPE gave a better cleanup efficiency than C18 cartridge and a higher recovery than Oasis HLB cartridge. Finally, the method of liquid chromatography–tandem mass spectrometry coupled with molecular-imprinted solid-phase extraction was validated in real samples including lobster, duck, honey, and egg. The spiked recoveries of TCs ranged from 94.51% to 103.0%. The limits of detection were in the range of 0.1–0.3 μg kg⁻¹. Chromatograms obtained by direct injection of the spiked egg extracts (5 × 10^-3 mmol L⁻¹) and purification with MISPE     

Small-volume fiber-optic evanescent-wave absorption sensor for nitrite determination

A novel small-volume fiber-optic evanescent-wave absorption sensor based on the Griess–Ilosvay reaction has been developed and evaluated for nitrite determination. The sensor was constructed by inserting a decladded optical fiber into a transparent capillary to form an annular column microchannel. The Evanescent wave (EW) field produced on the optical fiber core surface penetrated into the surrounding medium and interacted with the azo dye, which was generated by the reaction of nitrite and nitrite-sensitive reagents. The detector was designed to be parallel to the axis of the optical fiber. The defined absorbance was linear with the concentration of nitrite in the range from 0.05 to 10 mg L⁻¹, and the detection limit was 0.02 mg L⁻¹ (3σ) with the relative standard deviation (RSD) of 2.6% (n = 8). The present sensor was successfully used to determine nitrite in real samples of mineral water, tap water, rain water, and seawater. The results were consistent with the data obtained by standard spectrophotometric method, showing potential of the proposed sensor for practical application.      

Photofragmentation of nitro-based explosives with chemiluminescence detection

A simple, fast, reliable, sensitive and potentially portable explosive detection device was developed employing laser photofragmentation (PF) followed by heterogeneous chemiluminescence (CL) detection. The PF process involves the release of NO_{x(x = 1,2)} moieties from explosive compounds such as TNT, RDX, and PETN through a stepwise excitation–dissociation process using a 193 nm ArF laser. The NO_{x(x = 1,2)} produced upon PF is subsequently detected by its CL reaction with basic luminol solution. The intensity of the CL signal was detected by a thermoelectrically cooled photomultiplier tube with high quantum efficiency and negligible dark current counts. The system was able to detect trace amounts of explosives in various forms in real time under ambient conditions. Detection limits of 3 ppbv for PETN, 2 ppbv for RDX, and 34 ppbv for TNT were obtained. It was also demonstrated that the presence of PETN residue within the range of 61 to 186 ng/cm² can be detected at a given signal-to-background ratio of 10 using a few microjoules of laser energy. The technique also demonstrated its potential for the direct analysis of trace explosive in soil. An LOD range of 0.5–4.3 ppm for PETN was established, which is comparable to currently available techniques. Figure Photofragmentation–chemiluminescence detector     

Determination of endpoint of procedure for radix rehmanniae steamed based on ultraviolet spectrophotometry combination with continuous wavelet transform and kernel independent component analysis

A method for determination of the endpoint of the procedure for radix rehmanniae steamed was proposed based on UV spectrophotometry combination with continuous wavelet transform and kernel independent component analysis (UV-CWT-KICA). In the proposed method, the raw UV spectra of the rehmanniae samples during steamed procedure were measured. The raw UV spectral data were firstly pretreated by CWT for elimination of the noise signal and enrichment of the spectral resolution, then the independent components (ICs) were estimated from the mixed CWT coefficient matrix. The results show that the ICs are chemical significance with their relative concentrations gradually decreasing or increasing during the first steamed period, and the endpoint of the steamed procedure can be determined by inspection of the relative concentration profiles, at which the ICs should be approached maximum or minimum. Furthermore, the estimated ICs of rehmanniae samples from different areas or with different grades are similar, and the relative concentration of the similar ICs in different groups are increasing or decreasing before the first 14 h, and nearly steady or some decreasing after 16 h. Based on the variations of the relative concentration profiles of the ICs, the endpoint of the steamed procedure can be determined as 15 h, while that determined by sensory analysis is 14-20 h. The proposed UV-CWT-KICA method can avoid the higher deviations of the endpoints that were determined by sensory analysis. It provides an alternative approach for determination of the endpoint of the procedure for processing traditional Chinese medicine (TCM).     

Intensified biochip system using chemiluminescence for the detection of Bacillus globigii spores

This paper reports the first intensified biochip system for chemiluminescence detection and the feasibility of using this system for the analysis of biological warfare agents is demonstrated. An enzyme-linked immunosorbent assay targeting Bacillus globigii spores, a surrogate species for Bacillus anthracis, using a chemiluminescent alkaline phosphatase substrate is combined with a compact intensified biochip detection system. The enzymatic amplification was found to be an attractive method for detection of low spore concentrations when combined with the intensified biochip device. This system was capable of detecting approximately 1 × 10⁵ Bacillus globigii spores. Moreover, the chemiluminescence method, combined with the self-contained biochip design, allows for a simple, compact system that does not require laser excitation and is readily adaptable to field use. Figure Schematic diagram of the miniature biochip detection system     

The size distribution of 'gold standard' nanoparticles

The spherical gold nanoparticle reference materials RM 8011, RM 8012, and RM 8013, with a nominal radius of 5, 15, and 30 nm, respectively, have been available since 2008 from NIST. These materials are recommended as standards for nanoparticle size measurements and for the study of the biological effects of nanoparticles, e.g., in pre-clinical biomedical research. We report on determination of the size distributions of these gold nanoparticles using different small-angle X-ray scattering (SAXS) instruments. Measurements with a classical Kratky type SAXS instrument are compared with a synchrotron SAXS technique. Samples were investigated in situ, positioned in capillaries and in levitated droplets. The number-weighted size distributions were determined applying model scattering functions based on (a) Gaussian, (b) log-normal, and (c) Schulz distributions. The mean radii are 4.36 ± 0.04 nm (RM 8011), 12.20 ± 0.03 nm (RM 8012), and 25.74 ± 0.27 nm (RM 8013). Low polydispersities, defined as relative width of the distributions, were detected with values of 0.067 ± 0.006 (RM 8011), 0.103 ± 0.003, (RM 8012), and 0.10 ± 0.01 (RM 8013). The results are in agreement with integral values determined from classical evaluation procedures, such as the radius of gyration (Guinier) and particle volume (Kratky). No indications of particle aggregation and particle interactions—repulsive or attractive—were found. We recommend SAXS as a standard method for a fast and precise determination of size distributions of nanoparticles.      

Circular dichroism thermal lens microscope in the UV wavelength region (UV-CD-TLM) for chiral analysis on a microchip

We have developed a circular-dichroism thermal lens microscope for UV wavelengths (UV-CD-TLM), for the first time, to realize sensitive chiral analysis on a microchip. Quasi-continuous-wave phase modulation of a pulsed UV laser was used to generate left-circularly polarized light and right-circularly polarized light and to detect the generated TL signal amplitude and phase with a lock-in amplifier. The amplitude and phase were used to determine the concentration and chirality, respectively, of a sample. The basic principle of UV-CD-TLM for chiral analysis on a microchip was verified by measuring aqueous solutions of optically active camphorsulfonic acids (CSA). Lower limits of detection (LOD) were calculated at S/N = 2 and were 8.7 × 10⁻⁴ mol L⁻¹ (ΔA = 5.2 × 10⁻⁶ Abs.) for (+)-CSA and 8.4 × 10⁻⁴ mol L⁻¹ (ΔA = 5.0 × 10⁻⁶ Abs.) for (−)-CSA. In terms of number of molecules, LODs for UV-CD-TLM were calculated to be 8.7 fmol and 8.4 fmol, respectively. This is at least three orders of magnitude lower than previously obtained. The applicability of UV-CD-TLM for chiral analysis on a microchip was verified. Figure Sensitive chiral analysis by thermal lens microscope (TLM)     

Incubation type Si-based planar ion channel biosensor

A new planar-type ion channel biosensor with the function of cell culture has been fabricated using silicon on an insulator substrate as the sensor chip material. Coating of the sensor chip with fibronectin was essentially important for cell incubation on the chip. Although the seal resistance was quite low (∼7 MΩ) compared with the pipette patch-clamp gigaohm seal, the whole-cell channel current of the transient receptor potential vanilloid type 1 (TRPV1) channel expressing HEK293 cells was successfully observed, with a good signal-to-noise ratio, using capsaicin as a ligand molecule. Figure A new planer type ion channel biosensor with function of cell culture is fabricated using the silicon on insulator substrate as the sensor chip material. The coating of the sensor chip by the fibronectin was essentially important for the cell incubation on the chip. Whole cell channel current of TRPV1 channel was successfully observed using capsaicin as a ligand molecule with good signal to noise.     

Highly efficient analysis of underivatized carbohydrates using monolithic-silica-based capillary hydrophilic interaction (HILIC) HPLC

A polyacrylamide (PAAm)-modified monolithic silica capillary column of increased phase ratio, 200T-PAAm, for hydrophilic interaction liquid chromatography (HILIC) was prepared. The column showed high separation efficiency, with a theoretical plate height H = 7–20 μm at a linear velocity, u = 1–7 mm/s. From a kinetic plot analysis, it was expected that the monolithic column could provide three times faster separation than particle-packed HILIC columns under a pressure limit at 20 MPa. HILIC coupled with electrospray ionization (ESI)–mass spectrometry (HILIC-ESI-MS) using the 200T-PAAm column was employed for the analysis of underivatized carbohydrates to achieve fast and efficient separations of mixtures containing mono-, di-, and trisaccharides within 5 min. Under single MS full scan mode, 200 pg of oligosaccharides was detected by the system. The limit of detection (LOD) of the LC-ESI-MS/MS system was determined using selected reaction monitoring (SRM) to be as low as 3.2 ng/mL (attomol level) for nonreducing saccharides. The system was successfully applied to the detection of disaccharides in extracts of plant, such as corn, soybean, and Arabidopsis thaliana. Figure HILIC-ESI-MS provides a high-efficiency separation and sensitive detection of underivatized carbohydrate oligomers, e.g., the homologs of glucose (1) up to maltoheptaose (7)     

Towards an unbiased metabolic profiling of protozoan parasites: optimisation of a Leishmania sampling protocol for HILIC-orbitrap analysis

Comparative metabolomics of Leishmania species requires the simultaneous identification and quantification of a large number of intracellular metabolites. Here, we describe the optimisation of a comprehensive metabolite extraction protocol for Leishmania parasites and the subsequent optimisation of the analytical approach, consisting of hydrophilic interaction liquid chromatography coupled to LTQ-orbitrap mass spectrometry. The final optimised protocol starts with a rapid quenching of parasite cells to 0 °C, followed by a triplicate washing step in phosphate-buffered saline. The intracellular metabolome of 4 × 10⁷ parasites is then extracted in cold chloroform/methanol/water 20/60/20 (v/v/v) for 1 h at 4 °C, resulting in both cell disruption and comprehensive metabolite dissolution. Our developed metabolomics platform can detect approximately 20% of the predicted Leishmania metabolome in a single experiment in positive and negative ionisation mode.      

Automated extraction of direct,reactive, and vat dyes from cellulosic fibers for forensic analysis by capillary electrophoresis

Systematic designed experiments were employed to find the optimum conditions for extraction of direct, reactive, and vat dyes from cotton fibers prior to forensic characterization. Automated microextractions were coupled with measurements of extraction efficiencies on a microplate reader UV–visible spectrophotometer to enable rapid screening of extraction efficiency as a function of solvent composition. Solvent extraction conditions were also developed to be compatible with subsequent forensic characterization of extracted dyes by capillary electrophoresis with UV–visible diode array detection. The capillary electrophoresis electrolyte successfully used in this work consists of 5 mM ammonium acetate in 40:60 acetonitrile–water at pH 9.3, with the addition of sodium dithionite reducing agent to facilitate analysis of vat dyes. The ultimate goal of these research efforts is enhanced discrimination of trace fiber evidence by analysis of extracted dyes. Figure Fitted absorbance response surface for extraction of a direct dye, C. I. yellow 58, using a ternary solvent system.     

GC–MS analysis of low-molecular-weight dicarboxylic acids in atmospheric aerosol: comparison between silylation and esterification derivatization procedures

This paper describes methods for the determination of low-molecular-weight (LMW) dicarboxylic acids in atmospheric aerosols as important chemical tracers for source apportionment of aerosol organics and for studying atmospheric processes leading to secondary organic aerosol formation. The two derivatization procedures most widely used in GC analysis of dicarboxylic acids were compared: esterification using BF₃/alcohol reagent and silylation using N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA). The advantages and drawbacks of the two methods are investigated and compared in terms of (1) precision and accuracy of the results and (2) sensitivity and detection limit of the procedure. The comparative investigation was performed on standard solutions containing target C₃–C₉ dicarboxylic acids and on experimental particulate matter (PM) samples. Attention was focused on low-volume sampling devices that collect small amounts of sample for organic speciation. The results show that, overall, both the techniques appear suitable for the analysis of LMW dicarboxylic acids in atmospheric aerosols since they provide low detection limits (≤4 ng m⁻³) and satisfactory reproducibility (RSD% ≤ 15%). Between them, BSTFA should be the reagent of choice under the most limiting conditions of PM filters collected by low-volume air samplers: It provides determination of all the target C₃–C₉ dicarboxylic acids with lower detection limits (≤2 ng m⁻³) and higher reproducibility (RSD% ≤ 10%)