Metabolism-triggered sensor array aided by machine learning for rapid identification of pathogens.

Chemical-nose strategy has achieved certain success in the discrimination and identification of pathogens. However, this strategy usually relies on non-specific interactions, which are prone to be significantly disturbed by the change of environment thus limiting its practical usefulness. Herein, we present a novel chemical-nose sensing approach leveraging the difference in the dynamic metabolic variation during peptidoglycan metabolism among different species for rapid pathogen discrimination. Pathogens were first tethered with clickable handles through metabolic labeling at two different acidities (pH = 5 and 7) for 20 and 60 min, respectively, followed by click reaction with fluorescence up-conversion nanoparticles to generate a four-dimensional signal output. This discriminative multi-dimensional signal allowed eight types of model bacteria to be successfully classified within the training set into strains, genera, and Gram phenotypes. As the difference in signals of the four sensing channels reflects the difference in the amount/activity of enzymes involved in metabolic labeling, this strategy has good anti-interference capability, which enables precise pathogen identification within 2 h with 100% accuracy in spiked urinary samples and allows classification of unknown species out of the training set into the right phenotype. The robustness of this approach holds significant promise for its widespread application in pathogen identification and surveillance.

Multiplex Profiling of Biomarker and Drug Uptake in Single Cells Using Microfluidic Flow Cytometry and Mass Spectrometry.

To perform multiplex profiling of single cells and eliminate the risk of potential sample loss caused by centrifugation, we developed a microfluidic flow cytometry and mass spectrometry system (µCytoMS) to evaluate the drug uptake and induced protein expression at the single cell level. It involves a microfluidic chip for the alignment and purification of single cells followed by detection with laser-induced fluorescence (LIF) and inductively coupled plasma mass spectrometry (ICP-MS). Biofunctionalized nanoprobes (BioNPs), conjugating ∼3000 6-FAM-Sgc8 aptamers on a single gold nanoparticle (AuNP) (Kd = 0.23 nM), were engineered to selectively bind with protein tyrosine kinase 7 (PTK7) on target cells. PTK7 expression induced by oxaliplatin (OXA) uptake was assayed with LIF, while ICP-MS measurement of 195Pt revealed OXA uptake of the drug in individual cells, which provided further in-depth information about the drug in relation to PTK7 expression. At an ultralow flow of ∼0.043 dyn/cm2 (20 µL/min), the chip facilitates the extremely fast focusing of BioNPs labeled single cells without the need for centrifugal purification. It ensures multiplex profiling of single cells at a throughput speed of 500 cells/min as compared to 40 cells/min in previous studies. Using a machine learning algorithm to initially profile drug uptake and marker expression in tumor cell lines, µCytoMS was able to perform in situ profiling of the PTK7 response to the OXA at single-cell resolution for tests done on clinical samples from 10 breast cancer patients. It offers great potential for multiplex single-cell phenotypic analysis and clinical diagnosis.

Chemical Nose Strategy with Metabolic Labeling and "Antibiotic-Responsive Spectrum" Enables Accurate and Rapid Pathogen Identification.

The worldwide antimicrobial resistance (AMR) dilemma urgently requires rapid and accurate pathogen phenotype discrimination and antibiotic resistance identification. The conventional protocols are either time-consuming or depend on expensive instrumentations. Herein, we demonstrate a metabolic-labeling-assisted chemical nose strategy for phenotyping classification and antibiotic resistance identification of pathogens based on the "antibiotic-responsive spectrum" of different pathogens. d-Amino acids with click handles were metabolically incorporated into the cell wall of pathogens for further clicking with dibenzocyclooctyne-functionalized upconversion nanoparticles (DBCO-UCNPs) in the presence/absence of six types of antibiotics, which generates seven-channel sensing responses. With the assistance of machine learning algorithms, eight types of pathogens, including three types of antibiotic-resistant bacteria, can be well classified and discriminated in terms of microbial taxonomies, Gram phenotypes, and antibiotic resistance. The present metabolic-labeling-assisted strategy exhibits good anti-interference capability and improved discrimination ability rooted in the unique sensing mechanism. Sensitive identification of pathogens with 100% accuracy from artificial urinary tract infection samples at a concentration as low as 105 CFU/mL was achieved. Pathogens outside of the training set can also be discriminated well. This clearly demonstrated the potential of the present strategy in the identification of unknown pathogens in clinical samples.

Interaction of Cellular Uptake of Nanosilver and Metallothionein Stress Expression Elucidated by 2D Single-Cell Analyses Based on LIF and ICP-MS.

The exploration of cytology mechanisms of nanosilver uptake, toxicity, and detoxification has become an important issue due to its widespread applications. Previous studies have shown differences in the toxic response of mammalian cells to nanosilver. However, the analysis results based on cell populations ignore the impact of cell uptake heterogeneity on the expression of associated stress proteins and cellular physiological activities. In this respect, this work investigated the interaction between silver uptake and metallothionein (MT) expression in individual cells. In addition, we have also preliminarily elucidated the sensitivity variation to AgNPs by using five cell lines, e.g., LX-2, HepG-2, SK-HEP-1, Huh-7, and MDA-MB-231, by adopting a two-dimensional (2D) high-throughput single-cell analysis platform coupling laser-induced fluorescence (LIF) and inductively coupled plasma mass spectrometry (ICP-MS). We developed a 2D data analysis method for one-to-one unification of fluorescence-mass spectrometry signals corresponding to a specific single cell. It indicated that there is no obvious correlation between cellular silver uptake and cell size, and the low MT expression of cells is more sensitive to silver nanoparticles. For each cell line, significant heterogeneity in MT expression was observed. This provides important information for understanding the potential heterogeneous effects of nanosilver on mammalian biological systems. Overall, detoxified cells are more tolerant to nanosilver and normal cells are more tolerant than cancer cells.

Single Cell Phenotypic Analysis for Cancer Stem Cell Identification by Dual-Isotope ICP-QMS.

Single cell phenotypic analysis is significant for clinical diagnosis, treatment, and prognosis of cancer. Accurate differentiation of cancer stem cell (CSC) subpopulations from a large number of cancer cells may become a cancer surveillance tool and provide important implications for the development of new CSC-targeted therapy strategies. Herein, we report a new approach based on dual-isotope inductively coupled plasma quadrupole mass spectrometry (ICP-QMS) for single cell phenotypic analysis. High-throughput single cell sampling was achieved by a spiral channel microfluidic chip for cell focusing and alignment, and single cell analysis was performed with time-resolved ICP-QMS by identifying the highly specific probes. This enables the monitoring of two surface protein markers (EpCAM and MUC1) of three cell types, i.e., HeLa, MCF-7, and HepG2, at single cell level. The analysis of breast cancer stem cells further confirmed its capability in distinguishing rare cell phenotypes. The present study provides promising possibilities for adopting ICP-QMS in biomedical investigations in terms of cell typing, stemness identification of tumor cells, and cell heterogeneity analysis.

Two-Dimensional Multi-parameter Cytometry Platform for Single-Cell Analysis.

A 2D flow cytometry platform, known as CytoLM Plus, was developed for multi-parameter single-cell analysis. Single particles or cells after hydrodynamic alignment in a microfluidic unit undergo first-dimension fluorescence and side scattering dual-channel optical detection. They were thereafter immediately directed to ICP-MS by connecting the microfluidic unit with a high-efficiency nebulizer to facilitate the second-dimension ICP-MS detection. Flow cytometry measurements of fluorescent microspheres evaluated the performance of CytoLM Plus for optical detection. 6434 fluorescence bursts were observed with a valid signal proportion as high as 99.7%. After signal unification and gating analysis, 6067 sets of single-particle signals were obtained with 6.6 and 6.2% deviations for fluorescence burst area and height, respectively. This is fairly comparable with that achieved by a commercial flow cytometer. Afterward, CytoLM Plus was evaluated by 2D flow cytometry measurement of Ag+-incubated and AO-stained MCF-7 cells. A program for 2D single-cell signal unification was developed based on the algorithm of screening in lag time window. In the present case, a lag time window of -4.2 ± 0.09 s was determined by cross-correlation analysis and two-parameter optimization, which efficiently unified the concurrent single-cell signals from fluorescence, side scattering, and ICP-MS. A total of 495 sets of concurrent 2D signals were screened out, and the statistical analysis of these single-cell signals ensured 2D multi-parameter single-cell analysis and data elucidation.

Data analysis for the characterization of nanoparticles with single particle inductively coupled plasma mass spectrometry: From microsecond to millisecond dwell times.

Single particle-inductively coupled plasma-mass spectrometry (SP-ICP-MS) has become a powerful technique for the characterization of nanoparticles (NPs). However, the accuracy of the characterization of NPs by SP-ICP-MS is greatly affected by the data acquisition rate and the way of data processing. For SP-ICP-MS analysis, ICP-MS instruments typically apply microsecond to millisecond dwell times (10 µs-10 ms). Considering the duration of one nanoparticle event in the detector is 0.4-0.9 ms, NPs will show different data forms when working with microsecond and millisecond dwell times. In this work, the effects of dwell times from microsecond to millisecond (50 µs, 100 µs, 1 ms and 5 ms) on the data forms in SP-ICP-MS analysis are discussed. The data analysis and data processing for different dwell times is discussed in detail, including the measurement of transport efficiency (TE), the distinction of signal and background, the evaluation of diameter limit of detection (LODd) and the quantification of mass, size and particle number concentration (PNC) of NPs. This work provides data support for the data processing process and aspects to be considered in the characterization of NPs by SP-ICP-MS, which is expected to provide guidance and reference for researchers in SP-ICP-MS analysis.

Open flow cytometer with the combination of 3D hydrodynamic single cell focusing and confocal laser-induced fluorescence detection.

Flow cytometry is among the most powerful tools for single-cell analysis, while the high cost and mechanical complexity of the commercial instrumentation limit the applications in personalized single-cell analysis. For this issue, we hereby construct an open and low-cost flow cytometer. It is highly compact to integrate the functions of (1) single cell aligning by a lab-made modularized 3D hydrodynamic focusing device, and (2) fluorescence detection of the single cells by a confocal laser-induced fluorescence (LIF) detector. The ceiling cost of the entire hardware for the LIF detection unit and 3D focusing device is $ 3200 and $ 400 respectively. A sheath flow velocity of 150 µL/min produces a focused sample stream of 17.6 µm × 14.6 µm at sample flow of 2 µL/min, based on the LIF response frequency and the laser beam spot diameter. The assay performance of the flow cytometer was evaluated by characterizing fluorescent microparticles and acridine orange (AO) stained HepG2 cells, producing throughputs of 40.5/s and 6.2/s respectively. Favorable assay precision and accuracy were demonstrated by the agreement of frequency histogram with imaging analysis, and good Gaussian-like distributions of fluorescent microparticles and AO-stained HepG2 cells. Practically, the flow cytometer was successfully applied for the evaluation of ROS generation in single HepG2 cells.

[Determination of glutathione in cells by capillary electrophoresis-laser induced fluorescence].

Glutathione (GSH) is vital for oxidative stress resistance and heavy metals detoxification. It is significant to develop a sensitive and accurate quantitative GSH approach for the toxicity mechanism for studying heavy metals in cells. A high-sensitive capillary electrophoresis-laser induced fluorescence (CE-LIF) detection approach was proposed in this study to detect GSH content in cells. The approach employed HepG2 cells as an object and 2,3-naphthalenedicarboxaldehyde (NDA) with the active group of aromatic o-dialdehyde as a labeling reagent. The effects of buffer solution types, pH, additives on the GSH reaction rate with NDA, and the sensitivity of NDA-GSH were systematically investigated. The sensitivity of NDA-GSH and the reaction rate of GSH with NDA were compared in tris(hydroxymethyl)aminomethane (Tris) buffer solution at pH 7.4 or 9.2 and borate-Tris buffer solution at pH 9.2. The results revealed that the NDA-GSH sensitivity was the highest and the reaction rate of GSH and NDA was the fastest in borate buffer solution at pH 9.2. The effects of the four additives on the sensitivity of NDA-GSH were further compared. The best additive was revealed to be ß-cyclodextrin (ß-CD). GSH reacted with NDA to reach equilibrium within 5 min under the optimal experimental conditions, and the electrophoretic signal of NDA-GSH could be seen in 3 min. Quantitative analysis of GSH in HepG2 cells was performed using an external standard approach by determining a series of GSH standard solutions. The results revealed that the approach had a good linear relationship with the peak area vs. concentration (0.01-20.00 mmol/L) of GSH. The limit of detection (LOD) and limit of quantification (LOQ) of GSH were determined using signal-to-noise ratios of 3 (S/N=3) and 10 (S/N=10), which were 0.006 µmol/L and 0.020 µmol/L, respectively. The approach's spiked recoveries were 95.7%-112.6%, with relative standard deviations of the approach being 3.8%-5.0% (n=3). This approach offers high sensitivity, good stability, accuracy, and reliability. To study the relationship between the toxicity of arsenic and chromium on HepG2 cells and the content of GSH in HepG2 cells, the effects of arsenic and chromium with different valences on cell viability were analyzed. The results illustrated that the cytotoxicity of potassium dichromate (Cr(Ⅵ)) was the strongest. The variations of GSH content in HepG2 cells stimulated with arsenite (As(Ⅲ)), arsenate (As(Ⅴ)), chromium chloride (Cr(Ⅲ)), and Cr(Ⅵ) were analyzed by the proposed approach and analysis of intracellular GSH imaging. The results revealed that the stimulation group i. e. analyzed doses (low-dose 2 mg/L, high-dose 5 mg/L) of As(Ⅲ), As(Ⅴ), and Cr(Ⅲ) had no obvious effect on GSH content in HepG2 cells compared with the control group, whereas high-dose Cr(Ⅵ) can significantly reduce GSH content in HepG2 cells. Considering the analysis of cytotoxicity of As(Ⅲ), As(Ⅴ), Cr(Ⅲ), and Cr(Ⅵ), it shows that the content of GSH in HepG2 cells is related to cytotoxicity, and the content of GSH will decrease with the increase in cytotoxicity.

Intolerance of profligacy: an aptamer concentration gradient-tailored unicellular array for high-throughput biologics-mediated phenotyping.

In aptamer-based assay schemes, aptamer probes not labeled with biomarkers have to be eliminated before testing, which may lead to a tremendous waste of precious probes. We herein propose a microfluidics system integrating an aptamer concentration gradient generator (Apt-CGG) and a dual single-cell culturing array (D-SCA), termed Mi-Apt-SCA. This facilitates the precise construction of a nanoscale-gradient microenvironment and the high-throughput profiling of single-cell growth/phenotypes in situ with the minimal consumption of Apt-probe. Unlike previous snakelike mixers, the choreographed winding-ravined aptamer dual-spiral micromixer (Apt-WD-mixer) in Apt-CGG could allow thorough blending to generate linear concentration gradients of aptamer (quasi-non-Newtonian fluid) under the action of continuous fluidic wiggles and bidirectional Dean flow. In contrast to other trap-like systems, the mild vortex allows single-cell growth in an ultra-tender fluidic microenvironment using triple-jarless single-cell culture capsules (TriJ-SCCs) in D-SCA (shear stress: 3.43 × 10-5 dynes per cm2). The minimum dosage of aptamer probe required for exploring PDL1 protein expression in two hepatoma cell lines is only one-900th of that required by conventional protocols. In addition, this approach facilitated the profiling of ITF-ß/cisplatin-mediated single-cell/cell-cluster phenotypes.

Tracking cellular transformation of As(III) in HepG2 cells by single-cell focusing/capillary electrophoresis coupled to ICP-MS.

The cellular metabolism of metals is highly critical to elucidate their potential cytotoxicity or cell protection mechanism. In this work, an asymmetric serpentine microfluidic device (ASMD) with high sampling efficiency and excellent focusing performance was developed for single-cell focusing. ASMD coupling with ICP-MS ensures single-cell assay to provide the information for trivalent arsenic (As(III)) uptake by HepG2 cells, which reveals the heterogeneity of cellular arsenic distribution, and elucidates the arsenic elimination behaviors in single HepG2 cells. Further, the metabolism and transformation of As(III) in HepG2 cells was tracked by hyphenating capillary electrophoresis (CE) separation with ICP-MS. The results for single-cell analysis and arsenic elimination kinetics illustrated that the half-life of arsenic elimination is 0.9 ± 0.04 h with the elimination constant of 0.77 ± 0.03, i.e., 77% of accumulated As in HepG2 cells may be eliminated per hour. Moreover, arsenobetaine (AsB) was identified to be the main metabolite and biotransformation species of As in HepG2 cells. ASMD-ICP-MS and CE-ICP-MS are powerful for tracking the fate of metals or metal drugs in single cells to comprehensively understand their metabolic pathway and transformation behaviors.

Cryogenic Laser Ablation in a Rapid Cooling Chamber Ensures Excellent Elemental Imaging in Fresh Biological Tissues.

Laser ablation inductively coupled plasma mass spectrometry imaging of biologically significant targets largely relies on maintaining the original structures of samples. The temperature regulation capability of the ablation cell is crucial. Herein, a rapid cooling cryogenic sample cell (RCCSC) was developed. In the RCCSC chamber, the temperature reduces to -20 °C in 4 min with a minimum 10 h variation of ±0.1 °C at -26 °C. Improvements on the precision were achieved for the elements of interest in NIST 612 and spiked agarose gel under cryogenic conditions. The limits of detection improved by up to 1.57, 1.70, 3.26, and 1.33 fold for 63Cu, 66Zn, 57Fe, and 140Ce in agarose gel, respectively, were obtained under cryogenic conditions compared with those at room temperature. In a time period of testing (10 h), the cryogenic ablation maintains the native state of biological tissues with a high water content to ensure better elemental imaging by reducing thermal effects in ablation and suppressing evaporation of water. The rapid cooling cryogenic ablation significantly improves elemental imaging, as demonstrated by the imaging of various elements in coriander leaves. The present study may provide further insights into elemental distributions in fresh biological samples.

Characteristics of the Intestinal Microorganisms in Middle-Aged and Elderly Patients: Effects of Smoking.

Introduction: Smoking affects the occurrence and development of many diseases. We attempt to study the structure of intestinal flora in the middle-aged and elderly population as well as how smoking affects the intestinal flora. Methods: We collected population information, biochemical indicators, and patient feces from 188 middle-aged and elderly male patients, and their feces were tested for the 16S rRNA gene of intestinal flora. Results: We performed a cluster analysis on the intestinal structure of the included population and found that there was a significant difference in the number of smokers between each group (p = 0.011). Subsequently, the microbiological diversity analysis of current smokers and nonsmokers was carried out. The results indicated that there was a significant difference in species composition between the two groups (p = 0.029). Through the analysis on LEfSe differential bacteria, it was found that in current smoking patients, the abundances of the genus Bifidobacterium and the genus Coprobacillus were less, while the abundances of the genera Shigella, Paraprevotella, Burkholderia, Sutterella, Megamonas, and p-75-a5 under the family level of Erysipelotrichaceae were slightly high. We analyzed the correlation between the abundances of these eight different bacteria and clinical indicators. The results revealed the following: the abundance of the genus Bifidobacterium was negatively correlated with fasting blood glucose (r = -0.198, p = 0.006) and positively correlated with uric acid (r = 0.207, p = 0.004) and total bilirubin (r = 0.175, p = 0.017); Shigella bacteria were positively correlated with fasting blood glucose (r = 0.160, p = 0.028) and uric acid (r = 0.153, p = 0.036) levels; the genus Paraprevotella and BMI (r = -0.172, p = 0.018) are negatively correlated; the abundance of the genus Burkholderia was positively correlated with γ-glutamyltransferase (r = 0.146, p = 0.045) levels; Sutterella was correlated with fasting blood glucose (r = 0.143, p = 0.05) and creatinine level (r = -0.16, p = 0.027), which was positively correlated with fasting blood glucose and negatively correlated with creatinine. Conclusions: In middle-aged and elderly patients with cardiovascular disease, smoking can reduce the abundance of Bifidobacterium, while the abundances of some negative bacteria such as Burkholderia, Sutterella, and Megamonas increase.

Dual-Multivalent-Aptamer-Conjugated Nanoprobes for Superefficient Discerning of Single Circulating Tumor Cells in a Microfluidic Chip with Inductively Coupled Plasma Mass Spectrometry Detection.

The efficient recognition of circulating tumor cells (CTCs) with an aptamer probe confers numerous benefits; however, the stability and binding affinity of aptamers are significantly hampered in real biological sample matrices. Inspired by the efficient preying mechanism by multiplex tubing feet and endoskeletons of sea urchins, we engineered a superefficient biomimetic single-CTC recognition platform by conjugating dual-multivalent-aptamers (DMAs) Sgc8 and SYL3C onto AuNPs to form a sea urchin-like nanoprobe (sea urchin-DMA-AuNPs). Aptamers Sgc8 and SYL3C selectively bind with the biomarker proteins PTK7 and EpCAM expressed on the surface of CTCs. CTCs were captured with 100% efficiency, followed by sorting on a specially designed multifunctional microfluidic configuration, integrating a single-CTC separation unit and a hydrodynamic filtrating purification unit. After sorting, background-free analysis of biomarker proteins in single CTCs was undertaken with inductively coupled plasma mass spectrometry by measuring the amount of 197Au isotope in sea urchin-DMA-AuNPs. With respect to a single-aptamer nanoprobe/-interface, the dual-aptamer nanoprobe improves the binding efficiency by more than 200% (Kd < 0.35 nM). The microchip facilitates the recognition of single CTCs with a sorting separation rate of 93.6% at a flow rate of 60 µL min-1, and it exhibits 73.8 ± 5.0% measurement efficiency for single CTCs. The present strategy ensures the manipulation and detection of a single CTC in 100 µL of whole blood within 1 h.

Two-Dimensional Cytometry Platform for Single-Particle/Cell Analysis with Laser-Induced Fluorescence and ICP-MS.

A two-dimensional cytometry platform (CytoLM) with high sensitivity and high temporal resolution is developed for single-particle and single-cell sampling and analysis. First, a Dean flow-assisted vortex capillary cell sampling (VCCS) unit confines the sample stream in curved flow and drives to focus and align the particles or cells in a small probe volume. By coupling VCCS to a laser-induced fluorescence (LIF) detector with data acquisition and processing capability, a high-throughput single-particle/cell analysis system (VCCS-LIF) was established. The particle analysis throughput of 119.42/s and a detection recovery of 78.20 ± 1.75% were achieved at a density of 9.16 × 104/mL for fluorescent particles, and the cell analysis throughput is 48.20/s at a density of 1.5 × 105/mL. Second, the CytoLM platform is constructed by hyphenating VCCS-LIF with inductively coupled plasma mass spectrometry (ICP-MS). In the analysis of HepG2 cells by Ag+ incubation and AO staining, 10,760 fluorescence bursts and 3068 MS events were observed in 240 s. Invalid signals due to undispersed cells were controlled at 3.80% for LIF and 1.01% for MS, with a proportion of effective signal of >96.20%. After peak identification and integral processing of the original data, the statistical results including peak area, height, width, and spacing are obtained concurrently and the information on concentration and elemental quantification of single cells is evaluated. CytoLM facilitates high-throughput, multi-dimensional, and multi-parameter characterization of particles and cells, and it may provide vast potential in life science analysis.

Intracellular silver speciation by coupling capillary electrophoresis to ICP-MS integrating a high performance spiral flow spray chamber.

The study of silver species and their distribution/transformation in cell interior is of high significance for the elucidation of toxicology of silver nanoparticles (AgNPs). The intracellular speciation of dissolved Ag(I) and AgNPs was reported. The analytical platform integrated capillary electrophoresis (CE) to inductively coupled plasma-mass spectrometry (ICP-MS) incorporating a high efficiency interface and a high performance spiral flow spray chamber (SFSC). The interface and the SFSC provide a favorable detection limit of 87 ng L-1 for the dissolved Ag(I). Total silver content was quantified by ICP-MS subject to digestion of the cell lysate, and quantification of AgNPs was carried out by subtraction. The speciation of dissolved Ag(I) and AgNPs in culture medium and HepG2 cells was performed, with RSDs of <3% for relative peak area and <2% for migration time, as well as spiking recoveries of 93.8%-94.3% in opti-MEM and 92.7%-106.6% in cell lysate. The present study indicated higher solubility of AgNPs in the cell interior with respect to that in the culture medium, due to oxidative stress or acidic microenvironment in the cancer cells.

After enjoying curry: urine metabolism analysis of curcuminoids by microemulsion electrokinetic chromatography with laser-induced native fluorescence detection.

Considering pH-dependent fluorescence of curcuminoids, a microemulsion electrokinetic chromatographic (MEEKC) method was developed under acidic conditions for their separation and detection using laser-induced native fluorescence (LINF), so as to solve the analysis of urine metabolism for curcuminoids. The microemulsion composition was optimized by response surface methodology (RSM), and the effects of buffer pH and organic modifiers were systematically investigated. The optimal buffer for the separation of curcuminoids was chosen as follows: 2.8% (v/v) ethyl acetate, 80 mM SDS and 2.8% (v/v) n-butanol to form microemulsion, 28% (v/v) ethanol as organic modifier, and 20 mM phosphoric acid as electrolyte at pH 3.0. Under these conditions, four curcuminoids including curcumin, demethoxy curcumin (DMC), bisdemethoxy curcumin (BDMC) and demethyl curcumin (DEC) could be well separated within 18 min, and the detection limits (LOD, based on S/N=3) were calculated to be 71, 60, 22, and 147 pg mL-1, respectively. Combined with solid-phase extraction (SPE), the developed MEEKC-LINF method has been successfully applied to continuously monitor the curcuminoids and related metabolites in human urine collected from a healthy volunteer after oral administration of curry, testifying that this method has potential for evaluating the pharmacological activity of curcuminoids.

7-(Diethylamino)coumarin-3-carboxylic acid as derivatization reagent for 405 nm laser-induced fluorescence detection: A case study for the analysis of sulfonamides by capillary electrophoresis.

Diode lasers, especially 405 nm diode laser, as excitation sources offer new opportunities for laser-induced fluorescence (LIF) detection, but the lack of available derivatization reagents limits their widespread applications. Herein, a commercial fluorescent dye, 7-(diethylamino)coumarin-3-carboxylic acid (DEAC-C) was introduced as derivatization reagent and corresponding derivatization strategy for DEAC-C labeling sulfonamides was developed for 405 nm LIF detection. After systematic optimization, three sulfonamides including hydrochlorothiazide (HCTZ), chlorothiazide (CTZ) and chlortalidone (CTD) could be efficiently labeled by DEAC-C in the presence of cyanuric chloride and triethylamine (1.5%) in acetonitrile at 50 °C for 180 min. Based on the laboratory-built capillary electrophoresis-LIF system, a robust method was then proposed for the separation of DEAC-C labeled sulfonamides by micellar electrokinetic chromatography (MEKC) using the classic borax-SDS system. Under the optimized conditions, a baseline separation of three sulfonamides was achieved within 15 min, and the detection limits were determined to be 0.24, 0.29, and 0.23 nM for HCTZ, CTZ, and CTD, respectively. Furthermore, the developed MEKC-LIF method was validated using sulfonamide standards and spiked human urine sample and successfully applied for the analysis of three sulfonamides in complex pharmaceutical and physiological samples.

Fast screening of aflatoxins in dairy cattle feeds with CE-LIF method combined with preconcentration technique of vortex assisted low density solvent-microextraction.

Aflatoxin contamination in agricultural products poses a great threat to humans and livestock. The aim of this study was to establish a simple, rapid, highly sensitive, and inexpensive method for the simultaneous detection of aflatoxin B1 , B2 , G1 , and G2 in agricultural products. We used a vortex assisted low density solvent-microextraction (VALDS-ME) technique for sample preconcentration and sample detection was achieved with a CE-LIF method. Aflatoxins were separated in an uncoated fused-silica capillary with the MEKC mode and were excited by a 355 nm UV laser to produce native fluorescence for detection. The obtained LOD and LOQ for the four aflatoxins were in the range of 0.002-0.075 and 0.007-0.300 µg/L, respectively, and the analysis time was within 6.5 min. Using the established method, aflatoxins were screened in naturally contaminated dairy cattle feed samples including alfalfa, bran, and corn kernel. The result shows that the alfalfa and bran samples were contaminated with aflatoxins to varying degrees. Compared with other analytical techniques for aflatoxin screening in agricultural products, this CE-LIF method combined with VALDS-ME preconcentration technique is simple, rapid, highly efficient, and inexpensive.

Sensitive analysis of curcuminoids via micellar electrokinetic chromatography with laser-induced native fluorescence detection and mixed micelles-induced fluorescence synergism.

Curcuminoids, the major bioactive constituents of traditional medicine known as turmeric, have exhibited extensive therapeutic benefits. Excited by violet-blue light, curcuminoids can emit native fluorescence, making them possible to be detected with high sensitivity and specificity by laser-induced native fluorescence (LINF). Here, a commercial 445 nm laser diode was used as an excitation source to construct a confocal laser-induced fluorescence (LIF) detector and then a complete capillary electrophoresis (CE) system coupled with LIF detection was established. With three major curcuminoids, curcumin, demethoxy curcumin (DMC) and bisdemethoxy curcumin (BDMC) as target analytes, a micellar electrokinetic chromatography (MEKC) method was proposed using mixed micelles consisting of Triton X-100 and SDS to sensitize the native fluorescence of curcuminoids and enhance their separation efficiency. Fluorescence spectra revealed that the mixed micelles induced fluorescence synergism could enhance the signals of three curcuminoids by 77-, 57-, and 47-fold for curcumin, DMC, and BDMC. After systematic investigation, the optimal separation buffer for curcuminoids was chosen as follows: 20 mM Triton X-100, 20 mM SDS, 30% (v/v) methanol in 10 mM borax solution at pH 10.0. Under these conditions, a baseline separation of three curcuminoids was achieved within 10 min and the detection limits were found to be 4.1, 2.6, and 0.4 ng/mL for curcumin, DMC, and BDMC, respectively. Furthermore, the developed MEKC-LINF method was validated in terms of precision, linearity, accuracy and successfully applied for the determination of three curcuminoids in turmeric, medicinal turmeric liniment, curry seasoning, and human urine samples.