Simultaneous analysis of nine aromatic amines in mainstream cigarette smoke using online solid-phase extraction combined with liquid chromatography-tandem mass spectrometry.

A fully automated analytical method was developed and validated by this present study. The method was based on two-dimensional (2D) online solid-phase extraction liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) to determine nine aromatic amines (AAs) in mainstream smoke (MSS) simultaneously. As a part of validation process, AAs yields for 16 top-selling commercial cigarettes from China market were evaluated by the developed method under both Health Canada Intensive (HCI) and ISO machine smoking regimes. The gas phase of MSS was trapped by 25 mL 0.6 M hydrochloric acid solution, while the particulate phase was collected on a glass fiber filter. Then, the glass fiber pad was extracted with hydrochloric acid solution in an ultrasonic bath. The extract was analyzed with 2D online SPE-LC-MS/MS. In order to minimize the matrix effects of sample on each analyte, two cartridges with different extraction mechanisms were utilized to cleanup disturbances of different polarity, which were performed by the 2D SPE. A phenyl-hexyl analytical column was used to achieve a chromatographic separation. Under the optimized conditions, the isomers of p-toluidine, m-toluidine and o-toluidine, 3-aminobiphenyl and 4-aminobiphenyl, and 1-naphthylamine and 2-naphthylamine were baseline separated with good peak shapes for the first time. The limits of detection for nine AAs ranged from 0.03 to 0.24 ng cig-1. The recovery of the measurement of nine AAs was from 84.82 to 118.47%. The intra-day and inter-day precisions of nine AAs were less than 10 and 16%, respectively. Compared with ISO machine smoking regime, the AAs yields in MSS were 1.17 to 3.41 times higher under HCI machine smoking regime. Graphical abstract New method using online SPE-LC/MS/MS for analysis of aromatic amines in mainstream cigarette smoke.

Cationic Peptide Conjugation Enhances the Activity of Peroxidase-Mimicking DNAzymes.

Peroxidase-mimicking DNAzymes containing G-quadruplex structures are widely applied in chemistry as catalysts and signal amplification for biosensing. Enhancing the catalytic activity of these DNAzymes can therefore improve the performance of many catalysts and biosensors using them. In this work, we synthesized cationic peptide conjugates of peroxidase-mimicking DNAzymes, which were found to exhibit both enhanced peroxidase and oxidase activities up to 4-fold and 3-fold compared with the original DNAzymes, respectively. Further investigation suggested that the enhanced activity was ascribed to the stabilization of parallel DNA G-quadruplex structures and hemin binding by the cationic peptide covalently attached to the DNAzyme. Such a mechanism of activity enhancement was successfully utilized for biosensing applications with improved sensitivity and broadened target range. Hydrogen peroxide (H2O2) detection in K(+)-free solutions by the DNAzyme-peptide conjugate showed 2-fold sensitivity enhancement over the unmodified DNAzyme under the same condition, and the activity switch by target-induced cleavage of the DNAzyme-peptide conjugate was also used for the detection of caspase 3 protease with enzymatic amplification in homogeneous solutions.

A general approach for rational design of fluorescent DNA aptazyme sensors based on target-induced unfolding of DNA hairpins.

DNA aptazymes are allosteric DNAzymes activated by the targets of DNA aptamers. They take the advantages of both aptamers and DNAzymes, which can recognize specific targets with high selectivity and catalyze multiple-turnover reactions for signal amplification, respectively, and have shown their great promise in many analytical applications. So far, however, the available examples of DNA aptazyme sensors are still limited in utilizing only several DNAzymes and DNA aptamers, most likely due to the lack of a general and simple approach for rational design. Herein, we have developed such a general approach for designing fluorescent DNA aptazyme sensors. In this approach, aptamers and DNAzymes are connected at the ends to avoid any change in their original sequences, therefore enabling the general use of different aptamers and DNAzymes in the design. Upon activation of the aptazymes by the targets of interest, the rate of fluorescence enhancement via the cleavage of a dually labeled substrate by the active aptazymes is then monitored for target quantification. Two DNAzymes and two aptamers are used as examples for the design of three fluorescent aptazyme sensors, and they all show high selectivity and sensitivity for the detection of their targets. More DNA aptazyme sensors for a broader range of targets could be developed by this general approach as long as suitable DNAzymes and aptamers are used.

A ratiometric fluorescent chemosensor for Al³âº in aqueous solution based on aggregation-induced emission and its application in live-cell imaging.

A ratiometric fluorescent chemosensor 1 was developed for the detection of Al(3+) in aqueous solution based on aggregation-induced emmision (AIE). The chemosensor showed the fluorescence of its aggregated state and Al(3+)-chelated soluble state in the absence and in the presence of Al(3+), respectively, and resulted in a fluorescence ratio (I461/I537) response to Al(3+) in neutral aqueous solution at a detection limit as low as 0.29 µmol L(-1). The method was also highly selective to Al(3+) over other physiological relevant metal ions investigated in this study. Taking advantage of its AIE characteristics, the chemosensor was successfully applied on test papers for simple and rapid detection of Al(3+). Moreover, the application of 1 for the imaging of Al(3+) in living cells by ratiometric fluorescence changes was also achieved.

Simple and efficient method to purify DNA-protein conjugates and its sensing applications.

DNA-protein conjugates are very useful in analytical chemistry for target recognition and signal amplification. While a number of methods for conjugating DNA with proteins are known, methods for purification of DNA-protein conjugates from reaction mixture containing unreacted proteins are much less investigated. In this work, a simple and efficient approach to purify DNA-invertase conjugates from reaction mixture via a biotin displacement strategy to release desthiobiotinylated DNA-invertase conjugates from streptavidin-coated magnetic beads was developed. The conjugates purified by this approach were utilized for quantitative detection of cocaine and DNA using a personal glucose meter through structure-switching DNA aptamer sensors and competitive DNA hybridization assays, respectively. In both cases, the purified DNA-invertase conjugates showed better performance compared to the same assays using unpurified conjugates. The approach demonstrated here can be further expanded to other DNA and proteins to generate purified DNA-protein conjugates for analytical and other applications.

A new colorimetric strategy for monitoring caspase 3 activity by HRP-mimicking DNAzyme-peptide conjugates.

A new method for caspase 3 activity assay has been developed based on HRP-mimicking DNAzyme-peptide conjugates. The mechanism of detection was based on the specific cleavage of DEVD-peptides by active caspase 3 for recognition and the catalytic properties of HRP-mimicking DNAzymes for signal amplification. Under optimal conditions, the detection limit of caspase 3 was 0.89 nM. The proposed method was also successfully applied for the detection of caspase 3 in apoptosis cell lysates.

A ratiometric fluorescent probe for hydrophobic proteins in aqueous solution based on aggregation-induced emission.

A novel fluorescent probe 1 is reported here with ratiometric response to hydrophobic proteins (casein) or proteins with hydrophobic pockets (BSA, HSA) through hydrophobic interaction. Probe 1 underwent deprotonation in aqueous solution at pH 7.4 and emitted blue fluorescence at 436 nm. Upon the addition of BSA, HSA or casein, the aggregation-induced emission fluorescence of 1 at 518 nm was turned on. The fluorescence intensity ratio, I518/I436 was linearly related to the concentrations of these proteins. The detection limits for BSA, HSA and casein based on IUPAC (CDL = 3Sb m(-1)) were 16.2 µg mL(-1), 10.5 µg mL(-1) and 5.7 µg mL(-1), respectively.

Label-free catalytic and molecular beacon containing an abasic site for sensitive fluorescent detection of small inorganic and organic molecules.

In this work, two methods with complementary features, catalytic and molecular beacon (CAMB) and label-free fluorescent sensors using an abasic site, have been combined into new label-free CAMB sensors that possess advantages of each method. The label-free method using a dSpacer-containing molecular beacon makes CAMB more cost-effective and less interfering with the catalytic activity, while CAMB allows the label-free method to use true catalytic turnovers for signal amplifications, resulting in a new label-free CAMB sensor for Pb(2+) ion, with a detection limit of 3.8 nM while maintaining the same selectivity. Furthermore, by using CAMB to overcome the label-free method's limitation of requiring excess enzyme strands, a new label-free CAMB sensor using aptazyme is also designed to detect adenosine down to 1.4 µM, with excellent selectivity over other nucleosides.

A new coumarin-based fluorescence turn-on chemodosimeter for Cu2+ in water.

A highly selective and sensitive coumarin-based chemodosimeter 1 for Cu(2+) in water is reported in this work. 1 was designed and facilely synthesized by a one-step reaction with coumarin as a fluorophore and 2-picolinic acid as the binding moiety, which showed very week fluorescence in buffer solution, and its fluorescence was considerably enhanced by the addition of Cu(2+) at room temperature in 5 min. Mechanism study suggested that Cu(2+) promoted the hydrolysis of 1 via the catalytic sensing cycle, generating a highly fluorescent product 7-hydroxycoumarin with fluorescence signal greatly amplified. The probe exhibited remarkably selective fluorescence enhancement to Cu(2+) over other metal ions at 454 nm, with a detection limit of 35 nM Cu(2+). Under optimal condition, 1 was successfully used for the determination of Cu(2+) in fetal equine serum and two water samples.