•The SA-B-HRP nanocomplex is an effective signal amplification system in MIS.•MIS has been developed for detection of PCT and IL-6 simultaneously.•The LOD for PCT and IL-6 by MIS are 48.9 pg mL−1 and 1.0 pg mL−1, respectively.•MIS has successfully detected PCT and IL-6 simultaneously in serum samples.Simultaneous, sensitive and quantitative detection of biomarkers in infectious disease is crucial for guiding antimicrobial treatment and predicting prognosis. This work reported an ultrasensitive and quantitative microfluidic immunoassay combined with the streptavidin-biotin-peroxidase (SA-B-HRP) nanocomplex-signal amplification system (MIS) to detect two inflammatory biomarkers, procalcitonin (PCT, for discriminating bacterial infections from nonbacterial infections) and interleukin-6 (IL-6, for monitoring the kinetics of infectious disease) simultaneously. The amplification system was based on the one step self-assembly of SA and B-HRP to form the SA-B-HRP nanocomplex, which effectively amplified the chemiluminescent signals. The linear ranges for PCT and IL-6 detections by MIS were 250–1.28 × 105 pg mL−1 and 5–1280 pg mL−1, and the limit of detection (LOD) were 48.9 pg mL−1 and 1.0 pg mL−1, respectively, both of which were significantly improved compared with microfluidic immunoassays without amplification system (MI). More importantly, PCT and IL-6 in human serum could be simultaneously detected in the same run by MIS, which could greatly improve the detection efficiency and reduce the cost. Given the advantages of high sensitivity, multiplex and quantitative detection, MIS could be potentially applied for detection of biomarkers at low concentration in clinical diagnosis.Download high-res image (267KB)Download full-size image

An immunoassay has been developed for the determination of alpha-fetoprotein (AFP). It is based (a) on the use of a detection reaction mediated by manganese dioxide nanoparticles (MnO2 NPs) because their good stability at room temperature, and (b) on tyramine signal amplification (TSA). The MnO2 NPs acts as an artificial peroxidase that causes the conversion of TMB to give a colored product, and the tyramine-triggered reaction is used for signal amplification to improve the detection limit. Combined with immuno-magnetic separation and enrichment, the response of this AFP immunoassay is linear from 6.25–400 ng mL−1, with a detection limit of 22 pg mL−1 (S/N = 3). This immunoassay was successfully applied to the quantification of AFP in serum samples, and gave excellent accuracy compared with the clinical results from a local hospital. The LOD and stability of this assay are better than those of the standard horseradish peroxidase-based ELISA. The strategy presented here is conceived to have a wider scope in that it may be extended to various other immunoassays.

The authors describe a carbon dot (CD) based dual-emission ratiometric optical probe for the on-site visual and fluorometric determination of mercury(II) ions. The nanoparticle (NP) probe was obtained by covalently linking the blue emissive carbon dots to the surface of silica nanoparticles containing red-emissive quantum dots (QDs). The red emitting QDs in the silica matrix are inert to Hg(II) and provide a reliable and constant reference signal. They also reduce their toxicity and improve the optical and chemical stabilities, while the blue emission CDs are very sensitive to Hg(II). With increasing concentration of Hg(II), a solution containing the NP probe undergoes a continuous color change from light purple to red. This can be seen with bare eyes or detected instrumentally by measurement of fluorescence intensity under excitation/emission wavelengths of 350/453 and 658 nm. The probe exhibits high sensitivity to Hg(II), with a detection limit of 0.47 nM (at an S/N ratio of 3). This is much lower than the allowable level of mercury (10 nM, ~10 ppb) in drinking water set by the U.S. Environmental Protection Agency. For practical use, the probe was used to quantify Hg(II) in (spiked) tap water where it gave recoveries between 95 and 106% and relative standard deviations between 1.9 and 3.2%. The probe can also be applied in filter paper-based assays, and this paves the way to point-of-care pollution control. This ratiometric probe is nontoxic and easily operated, and therefore shows potential applications for rapid and low-cost visual identification of Hg(II).

•MNPs-poly-L-lysine brush (MPLLBs) has improved the binding capacity for protein.•MPLLBs is an attractive signal amplification system.•Enzyme-linked immunosorbent assay based MPLLBs (MELISA) improves the sensitivity.•The limit of detection of MELISA for Escherichia coli O157:H7 is 8 cfu/mL.We have developed a magnetic enzyme-linked immunosorbent assay (MELISA) based poly-L-lysine (PLL) mediated brushes on magnetic nanoparticles (MNPs) for detection of Escherichia coli O157:H7 in river water samples. In the MELISA, we couple PLL on the surface of magnetic nanoparticles to prepare the PLL brushes (MPLLBs). PLL with plentiful amine groups provides multi-binding sites to allow the binding of both horse radish peroxidase (HRP) and antibody (Ab) on the surface of the MPLLBs with high capacity. Compared with the conventional particles (the binding capacity of MNPs for HRP and Ab are 19 μg/mg and 16 μg/mg, respectively), MPLLBs have achieved an improvement of 43-fold and 24-fold in binding capacity for HRP (816 μg/mg, protein μg per mg of MPLLBs) and Ab (387 μg/mg), respectively. This multifunctional Ab-MPLLBs-HRP conjugate serves as not only an immune-carrier for magnetic enrichment but also an enzyme assembly for signal amplification system. Compared with the conventional ELISA (the detection of limit is 400 cfu/mL), MELISA shows enhanced sensitivity (8 cfu/mL) and shortened the analysis time (within 2 h) for the detection of Escherichia coli O157:H7 in river water sample, which provides an attractive candidate platform for the rapid and sensitive detection of pathogen in complex samples.Download high-res image (286KB)Download full-size image

•Binding modes of three alkaloids with KRAS G-quadruplex were comparatively probed by different spectroscopic methods.•Sanguinarine was more beneficial to maintain the parallel G-quadruplex structure.•Sanguinarine had a better binding capacity and a more effective antiproliferation activity on SW620 cells.•Binding property with KRAS G-quadruplex and antiproliferation effects of alkaloids were related to molecular structures.KRAS promoter can form G-quadruplex structure and regulate gene transcription. The drugs which can bind with G-quadruplex of KRAS promoter may be potential remedy for treatment of cancers associated with KRAS mutation. The interaction mechanism between the G-quadruplex of KRAS promoter and three isoquinoline alkaloids (jatrorrhizine, berberine and sanguinarine) has been investigated by UV-visible, fluorescence and circular dichroism spectroscopic methods. The results showed that the three alkaloids can form complexes with G-quadruplex KRAS promoter with the molecular ratio of 1:1, and the binding constants were (0.90 ± 0.16) × 106 L mol− 1, (0.93 ± 0.21) × 106 L mol− 1 and (1.16 ± 0.45) × 106 L mol− 1 for jatrorrhizine, berberine and sanguinarine. The absorption spectra, KI quenching and fluorescence anisotropy and polarization studies suggested jatrorrhizine and berberine interacted with G-quadruplex by not only end-stacking binding mode but also grooves or loops binding mode, while sanguinarine by end-stacking binding mode. Sanguinarine was more beneficial to maintain the stability and parallel conformation of KRAS promoter G-quadruplex. MTT assay was performed to evaluate antiproliferation effects of the three isoquinoline alkaloids on SW620 cells, and the antiproliferation effects of the three alkaloids were sanguinarine > berberine > jatrorrhizine. All the three alkaloids can bind with KRAS promoter G-quadruplex, and sanguinarine had the better binding property and antiproliferation effects on SW620 cells. The results obtained are meaningful to explore potential reagents targeting the parallel G-quadruplex structure of KRAS promoter for gene theraphy of colorectal carcinomas.

•Dual emission ratiometric fluorescence probe for detection of glucose was developed.•The developed probe can be utilized to quantitatively and visually detect glucose.•The probe was applied to detect glucose levels of beverages and biological samples.A novel dual emission ratiometric fluorescence probe for determination of glucose has been developed. The reference dye fluorescence isothiocyanate (FITC) has been encapsulated in the silica nanoparticles and then the red emission CdTe QDs were grafted on the surface of the silica particles to obtain the fluorescence probe. With glucose and dopamine as substrates, the glucose level was proportional to the fluorescence ratio change of above probe caused by dopamine oxidation, which was produced via bienzyme catalysis (glucose oxidase and horseradish peroxidase). The established approach was sensitive and selective, and has been applied to determine the glucose in beverage, urine and serum samples. The average recoveries of the glucose at various spiking levels ranged from 95.5% to 108.9% with relative standard deviations from 1.5% to 4.3%. The results provided a clue to develop sensors for rapid determination of the target analytes from complex matrices.

•Environmental friendly fluorescent FRET probe for detection of glucose was developed.•Natural pigment 3-hydroxyflavone can form FRET system with β-CD-modified ZnS QDs.•Hydrogen peroxide can oxidize 3-HF and lead to fluorescence quenching of the probe.•The probe was applied to determination of glucose in human serum and urine samples.•Developed approach was simple and with high sensitivity and selective for glucose.An environmental friendly fluorescence resonance energy transfer probe for determination of glucose has been developed. The natural pigment, 3-hydroxyflavone, can enter the hydrophobic cavity of beta-cyclodextrin functionalized ZnS quantum dots, forming a fluorescence resonance energy transfer system with beta-cyclodextrin modified ZnS quantum dots as energy donor and 3-hydroxyflavone as energy acceptors. Glucose can be oxidized with assistance of the glucose oxidase to quantitatively produce hydrogen peroxide, which could rapidly quench the fluorescence intensity of the 3-hydroxyflavone in the probe by catalysis of horseradish peroxidase. The probe was successfully applied to determination of glucose in human serum and urine samples, and the results indicated that the average spiking recoveries of glucose ranged from 102 to 113%, with relative standard deviations below 15%. The developed approach were simple and possessed high sensitivity and selective for glucose, and this work provides a new clue for developing fluorescent probe.

•The differentiation approach for carbonless copy papers (CCP) has been established.•Degradation products of crystal violet lactone (CVL) were identified by Q-TOF-MS.•Procedures for quantification of CVL and its degradation products in ink entries were developed.•Relationships of relative peak area versus aging time for CVL and its products were probed.•Dating method for the CCP samples has been explored and established.A method to differentiate between carbonless copy papers and to date the ink entries on those papers has been developed based on ultra-high-performance liquid chromatography and mass spectrometric methods. A total of 52 paper samples were distinguished and classified according to compositional differences reflected by their chromatograms, with a discrimination power as high as 99.3%. The degradation kinetics of a common dye, crystal violet lactone, was investigated, and a quantitative determination method for the dye and its decomposition products was established. The results showed that relative peak area for both the dye and its degradation products versus aging time exhibits satisfactory linearity, which was utilized to date the ink entries on the samples. Overall, this work details an approach that can discriminate between different samples on the basis of compositional differences and describes the degradation rate of the dye, which can be used to age and authenticate ink entries.

We develop a visual immunosensor for the one-step detection of Salmonella enterica by using gold nanoparticle triggered enzyme signal amplification and magnetic separation. This immunosensor shows an enhanced sensitivity compared with an enzyme-linked immunosorbent assay, without the involvement of any equipment, thus providing a promising platform for rapid and sensitive detection of pathogens.

We developed a magnetic relaxation switching immunosensor for the detection of salbutamol using a gold nanoparticles–streptavidin conjugate and magnetic nanoparticles. This immunosensor enables faster detection with enhanced sensitivity compared with enzyme-linked immunosorbent assay, providing a promising platform for rapid sensing of small molecules.

A novel water-soluble beta-cyclodextrin (β-CD)-functionalized ZnS quantum dot (QD)–neutral red (NR) fluorescence resonance energy transfer (FRET) probe for the selective determination of the concentration of megestrol acetate in river water has been developed. The water-soluble and low-toxicity β-CD-functionalized ZnS QDs were first synthesized, and their characterization was confirmed by transmission electron microscopy and infrared, UV-vis and fluorescence emission spectra. The NR molecule can enter the cavity of the β-CD anchored onto the surfaces of the ZnS QDs in its neutral form, forming the FRET probe. Compared with other steroid hormones, the probe can selectively recognize megestrol acetate at a lower concentration level. The possible underlying mechanism of the probe with nine steroid hormones was discussed in detail. The fluorescence quenching fractions of the probe presented a satisfactory linearity with the concentrations of megestrol acetate, and its limit of detection was calculated to be 0.0083 μM. Coupled with sample pretreatment procedures, the probe has been applied to the determination of megestrol acetate in river water. The average recoveries of megestrol acetate in the spiking levels of 0.001 μM–10 μM ranged from 97% to 110% with a relative standard deviation below 15%, which was similar to those for HPLC or MS techniques.

•A novel method for determination of ten steroid hormones has been developed.•Matrix-solid phase dispersion has been applied to pretreatment of the samples.•Procedures for matrix-solid phase dispersion have been investigated and optimised.•Developed method has high sensitivity and repeatability for the steroid hormones.•The method has been successfully applied to the analysis of real samples.An UPLC–MS/MS method for determination of ten steroid hormones in animal origin food has been developed with pretreatment of the samples by matrix solid-phase dispersion (MSPD). The MSPD conditions, including the dispersing sorbents, elution solvents, ratio of sorbent to sample and the volume of the elution solvent have been investigated and optimised, and the method has been evaluated and validated. The results showed that the developed method has satisfactory linearity between the MS/MS responses of the analytes and the concentration of the steroid hormones, and the limits of the detection can reach 0.01 μg/kg for most of the analytes. The spiking recoveries of the steroid hormones in chicken, pork, beef and sausage samples were between 76.8% and 98.7% with RSDs lower than 10%. The results demonstrated that the developed approach has high sensitivity and repeatability, and can rapidly determinate the trace residues of steroid hormones in complex food matrices.

The establishment of approaches for the differentiation of the ink entries of seals on paper can provide evidence to authenticate the related documents and can play a key role in judicial expertise. The identification and discrimination method for 38 red ink entries of seals on paper has been investigated using laser desorption ionization mass spectrometry (LDI-MS). Six dye components for the ink pastes of seals, Scarlet powder (SP), Bronze Red C (BR), Fast Red R (FR), Basic Violet 3 (BV3), Pigment Red 22 (PR22) and Pigment Red 112 (PR112), have been identified by their LDI-MS spectra, and the results have been confirmed by electrospray ionization quadruple-time of flight mass spectrometry (QTOF-ESI-MS/MS) and inductively coupled plasma atomic emission spectrometry (ICP-AES). The 38 ink entries were classified into six groups based on the presence or the absence of the pigments in their positive and negative LDI-MS spectra, and the discrimination power (DP) was calculated to be about 82%. The ink entries within each group were further differentiated from the relative peak areas (RPA) of the fragments for the pigments and the profile of their LDI-MS spectra, and thus the DP was increased to 98%. All the 38 ink entries could be discriminated (the DP was 100%), if including the contribution of unknown peaks. Compared with the results obtained by the FTIR and Raman methods, the established LDI-MS approach could provide more information of the dye components in the ink entries. The results showed that the developed LDI-MS method is powerful, sensitive and rapid and can directly differentiate the red ink entries of seals from paper substrates, thus offering a novel approach to judge the authenticity of documents.

The approaches for differentiation and dating of gel pen ink entries have been investigated by laser desorption ionization-time of flight mass spectrometry (LDI-TOF-MS) and high performance liquid chromatography-quadruple-time of flight mass spectrometry (HPLC-Q-TOF-MS). 45 kinds of black and blue gel pen ink entries were differentiated individually by the profiles of their LDI-MS spectra. The dye components in the black and blue ink entries have been identified by thin layer chromatography and HPLC-Q-TOF-MS methods. The degradation processes of the dye components in the ink entries under various aging conditions have been probed by LDI-MS approach. The results showed that the variations of relative intensities for the main dye components have a close relationship with aging time, and the degradation of the main dye components were significant under natural storage conditions, which can provide important evidences for dating of the ink entries on paper.Highlights► The gel pen ink entries have been differentiated by LDI-TOF-MS. ► The dye components of the ink entries have been identified by Q-TOF-MS. ► Degradation of the dye components in ink entries has been investigated by LDI-MS. ► Dating method for the ink entries were developed based on degradation of the dyes.

The interaction mechanism of 3,7-dihydroxyflavone (3,7-diHF) and human serum albumin (HSA) was investigated by fluorescence quenching, fluorescence enhancement, steady-state and time-resolved fluorescence emission and UV–vis absorption spectrometry. The binding site of 3,7-diHF on protein was determined by investigating the spectroscopic properties of 3,7-diHF–HSA complex at pH 7.4 and pH 3.5 individually, and confirmed by the site marker competitive experiments. The binding parameters of 3,7-diHF–HSA complex were estimated by fluorescence quenching experiments, and the data were in good agreement with the results obtained from fluorescence enhancement measurements. A remarkable increase in the fluorescence anisotropy values suggested that 3,7-diHF bound at a motional restricted pocket on HSA. The results indicated that 3,7-diHF, in anionic form, was bound within the hydrophobic pockets of the subdomain IIA of HSA (site I), and stabilised mainly by electrostatic force and ionic interactions. The binding mode of drug–protein was discussed based on above experimental results.Highlights► A novel method for determining the drug binding site on protein has been developed. ► Fluorescence quenching mechanism of HSA has been clarified with multiple approaches. ► Structural alterations of 3,7-diHF after binding to protein has been illustrated. ► Spectroscopic features of 3,7-diHF after binding to protein has been clarified. ► Binding mode of 3,7-diHF and HSA has been investigated and discussed.

Molecularly imprinted film with diphenolic acid (DPA) as dummy template molecule has been grafted on the surface of Mn-doped ZnS quantum dots (QDs) to develop a selective and sensitive sensor for rapid determination of tetrabromobisphenol A (TBBPA) in water and soils. The obtained DPA-MIP-QDs sensor has distinguished selectivity and high binding affinity to TBBPA. The fluorescence quenching fractions of the sensor presented a satisfactory linearity with the concentrations of TBBPA in the range of 0.1–100 μM, and its limit of detection can reach 0.015 μM. The sensor has been successfully applied to determine the TBBPA in water and soil samples, and the average recoveries of the TBBPA at various spiking levels ranged from 80.2% to 96.5% with relative standard deviation below 8.0%. The results provided a clue to develop sensors for rapid determination of hazardous materials from complex matrixes.

3,4′,7,8-Tetrahydroxyflavone (3,4′,7,8-tetraHF) is a kind of plant flavonol, and possesses anti-inflammatory, anti-nociceptive and anti-platelet effects. For understanding its pharmacology, the binding mechanism of 3,4′,7,8-tetraHF to a model protein, human serum albumin (HSA), was probed by fluorescence and UV absorption and mass spectrometric approaches. The binding affinities of the drug with the native HSA and its isomer were about 1.64 ± 0.54 × 105 L mol−1 and 0.68 ± 0.53 × 105 L mol−1, respectively. The UV absorption results showed that the hydroxyl groups for both the keto and enol forms of 3,4′,7,8-tetraHF were dissociated and the drug bound with the protein in anion. The fluorescence emission intensities and anisotropy of 3,4′,7,8-tetraHF were dramatically enhanced after interacting with the protein. The binding site of the drug on HSA was located on the motional restricted hydrophobic pocket of subdomain IIA, namely site I, and the drug–protein complex was mainly stabilized by electrostatic and ionic forces.Highlights► Structural features for the tautomers of 3,4′,7,8-tetraHF has been characterized. ► Complex of 3,4′,7,8-tetraHF and HSA has been determined by TOF-MS method. ► Spectroscopic property of drug after binding to various isomers of HSA was probed. ► The binding location of the drug on HSA was determined and validated. ► Binding mechanism of the drug and protein was explored with multiple approaches.

A novel approach for identification and determination of emulsion explosives with Span-80 (sorbitol mono-oleate) as the emulsifier and their postblast residues by gas chromatography–mass spectrometry (GC–MS) has been developed. 24 kinds of emulsion explosives collected have been processed by transesterification reaction with metholic KOH solution and the emulsifier has turned into methyl esters of fatty acids. From the peak area ratios of their methyl esters, most of these emulsion explosives can be differentiated. In order to detect the postblast residues of emulsion explosives, the sorbitols in the emulsifier Span-80 obtained after transesterification reaction have been further derivatized by silylation reaction with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS) as the derivatizing reagent. The derivatization conditions were optimized and the derivatives were determined by GC–MS. The results showed that the silylation derivatives of sorbitol and it isomers, combined with hydrocarbon compounds and methyl esters of fatty acids, were the characteristic components for identification of the emulsion explosives. The established approach was applied to analyze the postblast residues of emulsion explosives. It has been found that the method was sensitive and specific, especially when detecting the derivatives of sorbitol and its isomers by GC–MS in selecting ion mode. The information of the characteristic components can help probe the origin of the emulsion explosives and providing scientific evidences and clues for solving the crimes of the emulsion explosive explosion.

A novel approach for the identification and dating of the fountain pen ink entries on paper has been established by ion-pairing high-performance liquid chromatography (IP-HPLC). Twelve black and six red fountain inks have been collected, and their ink entries have been prepared by drawing lines on paper. The chromatographic conditions for separation of their dye components after extraction with solvents were optimized. Under the optimized conditions, the 18 fountain pen inks were differentiated individually by comparing the number of detectable main or minor dye components, and the relative peak intensities of each component. The ink entries were artificially and naturally aged, and the analysis results showed that the ink dye components were significantly decomposed when exposed to UV or fluorescent light compare to those of inks stored under natural condition. The changes of the relative peak height for the dye components were linearly related to the aging time, especially under natural aging conditions. The degradation characteristics of the dye components under different aging conditions provide scientific evidences for dating of the suspicious fountain pen ink entries on document.

Ion-pairing high performance liquid chromatography (IP-HPLC) was utilized to monitor the composition changes of blue gel pen ink entries on paper stored in different light conditions and natural environment. The chromatographic conditions were optimized by comparing the separation efficiencies of the blue gel pen inks using a series of ion-pairing reagents, including ammonium carbonate, ammonium acetate, triethylamine acetate, tributylamine acetate, tetrabutylammonium bromide and dihexylammonium acetate. It has been found that tributylamine acetate was a suitable ion-pairing reagent for separation of the inks on the common C18 column. The analysis results of the ink entries on paper in different aging conditions showed that the tendency of composition change in natural aging condition was similar with those in fluorescent light and UV light conditions, respectively. One main component dye of the blue gel pen ink, Acid Blue 9, and its degradation products were identified by ion-pairing high performance liquid chromatography coupled with electrospray tandem mass spectrometry. The results showed that the main degradation products originated from the Acid Blue 9. It gave a reasonable explanation for the changing rules of the relative content of the dyes in the blue gel pen ink. The results obtained can provide scientific evidences for dating of the blue gel pen ink entries on documents.

A novel approach for classification and dating of the black gel pen ink entries on document was developed based on ion-pairing high-performance liquid chromatography (IP-HPLC). Ninety-three black gel pens were collected and divided into two groups, dye-based and pigment-based, by preliminary solubility test. The chromatographic conditions for separation of the dye-based black gel pen inks were optimized and the dye components in inks were satisfactorily separated by using 40 mmol/L tetrabutylammonium bromide as ion-pairing reagent. According to the number and the chromatographic retention times of the main dye components, the 50 dye-based inks were categorized into four classes. The inks within a class can be further identified by the percentage of each dye component. The compositional changes of the dye components in the black gel pen ink entries on paper were investigated in light and natural aging conditions and it has been found that the dye components in the ink entries underwent obvious decomposition, and the decomposing extent of the dye components was related to the aging time. The results can provide scientific evidences for dating of the suspicious black gel pen ink entries on documents.

A rapid and simple method for determination and confirmation of the thyreostats in milk and urine has been developed. The samples were extracted and purified by matrix solid-phase dispersion (MSPD) with silica gel as the solid support, and the thyreostats including tapazole, 2-thiouracil, 6-methyl-2-thiouracil, 6-propyl-2-thiouracil and 6-phenyl-2-thiouracil have been separated and detected by gas chromatography–mass spectrometry in selected ion mode with dimethylthiouracil as internal standard after derivatization with pentafluorobenzylbromide and N-methyl-N-(trimethylsilyl)-trifluoroacetamide. The derivatization reaction was conducted in a strong basic condition, which can improve the derivatization yields of the thyreostats in the presence of the matrix. The relative abundances of the selected ions in the chromatograms of the derivatives were stable and consistent, and can be used for the confirmatory purpose. The average recoveries of the five thyreostatic compounds in milk and urine were above 70% in most cases with the relative standard deviations between 2.1 and 7.9%. The limit of detection was 0.0016 μg g−1 for the thyreostats except for that of tapazole, which was 0.004 μg g−1.