Protein triggered ordering transitions in poly (L-lysine)-coated liquid crystal emulsion droplets

Here, we report a simple and label-free methodology for real-time monitoring of adsorption of proteins such as bovine serum albumin (BSA), concanavalin A (ConA) (a lectin) and cathepsin D (CathD) (a tumour marker) on micrometer-sized poly (L-lysine) (PLL) functionalised liquid crystal (LC) droplets dispersed in aqueous phases. Earlier, we had demonstrated that PLL, a positively charged natural peptide, can induce homeotropic ordering of LCs at LC-aqueous interface, and thus PLL-adsorbed LC droplets showed radial director configuration. Herein, it was observed that subsequent non-specific adsorption of anionic proteins such as BSA, ConA and CathD can trigger a quick transition in director configuration of PLL-LC droplets (primarily dominated by electrostatic interactions) to pre-radial or bipolar, thus acting as a simple optical probe for detection of these proteins up to μg/mL of concentrations. Further, the detection limits for these proteins are found to vary (BSA<ConA<CathD) which follow the similar order as their anionic charges, thus suggesting the role of different binding affinities of protein-PLL in realising the director configuration of LC droplets. Overall, this study offers new pathways utilising ordering transition in LC droplets which will strengthen the principles to recognise biomolecular interactions for various interfacial and sensing applications.     

Label-free detection of proteins from dried-suspended droplets using surface enhanced Raman scattering

A simple sample preparation method to obtain rich and reproducible surface-enhanced Raman scattering (SERS) spectra from proteins regardless of their surface properties and dimensions for label-free detection and identification is reported. The method uses colloidal silver nanoparticles (AgNPs) as substrates and is based on suspending the droplet of a mixture containing AgNPs and proteins from a hydrophobic surface. Drying the droplet at this suspended configuration allows the accumulation and packing of AgNPs and protein molecules in the middle of the droplet area rather than getting jammed at the edges of drying droplets as the solvent evaporates. A detection limit of down to 0.05 μg mL(-1) for some of the model proteins used in this study is obtained with this simple approach. The advantage of this method is its simplicity and improved sensitivity over other approaches reported in the literature.     

用水溶性四苯基乙烯基荧光探针检测ctDNA

设计合成了一种水溶性的四苯基乙烯(TPE)衍生物TPEDPyMe,研究了该化合物的吸收和发射特性,发现TPEDPyMe具有聚集诱导发光(AIE)性能.在pH值为7.2的三羟甲基氨基甲烷-盐酸(tris-HCl)缓冲溶液中用TPEDPyMe检测小牛胸腺DNA(ctDNA)时观察到,随着ctDNA的浓度从0μg/mL增大到...     

A homogeneous assay for protein analysis in droplets by fluorescence polarization

We present a novel homogeneous (“mix‐incubate‐read”) droplet microfluidic assay for specific protein detection in picoliter volumes by fluorescence polarization (FP), for the first time demonstrating the use of FP in a droplet microfluidic assay. Using an FP‐based assay we detect streptavidin concentrations as low as 500 nM and demonstrate that an FP assay allows us to distinguish droplets containing 5 μM rabbit IgG from droplets without IgG with an accuracy of 95%, levels relevant for hybridoma screening. This adds to the repertoire of droplet assay techniques a direct protein detection method which can be performed entirely inside droplets without the need for labeling of the analyte molecules.     

Liquid crystal-based immunoassay for detecting human serum albumin

Human serum albumin (HSA) is the most abundant protein in human blood plasma. It is commonly used as a biomarker in urine samples to identify the chronic kidney disease caused by high blood pressure or diabetes. In our research, a thin layer of liquid crystals (LCs) is used as a readout system for developing an immunoassay that reports the presence of HSA in the aqueous solution with optical signals. The detection principle of this assay is based on the variation of surface density of protein upon the specific binding of HSA on an anti-HSA immobilized surface, which leads a dark-to-bright transition of LC images under cross-polarizers. Our results show that the LC-based immunoassay can detect HSA at a concentration of 50 μg/mL. By using the slide with immobilized anti-HSA in array format, the concentration level of HSA can be simply determined by the number of LC spot shown on the slide.     

Gold nanoparticle-based fluorescence immunoassay for malaria antigen detection

The development of rapid detection assays for malaria diagnostics is an area of intensive research, as the traditional microscopic analysis of blood smears is cumbersome and requires skilled personnel. Here, we describe a simple and sensitive immunoassay that successfully detects malaria antigens in infected blood cultures. This homogeneous assay is based on the fluorescence quenching of cyanine 3B (Cy3B)-labeled recombinant Plasmodium falciparum heat shock protein 70 (PfHsp70) upon binding to gold nanoparticles (AuNPs) functionalized with an anti-Hsp70 monoclonal antibody. Upon competition with the free antigen, the Cy3B-labeled recombinant PfHsp70 is released to solution resulting in an increase of fluorescence intensity. Two types of AuNP-antibody conjugates were used as probes, one obtained by electrostatic adsorption of the antibody on AuNPs surface and the other by covalent bonding using protein cross-linking agents. In comparison with cross-linked antibodies, electrostatic adsorption of the antibodies to the AuNPs surfaces generated conjugates with increased activity and linearity of response, within a range of antigen concentration from 8.2 to 23.8 μg.mL(-1). The estimated LOD for the assay is 2.4 μg.mL(-1) and the LOQ is 7.3 μg.mL(-1). The fluorescence immunoassay was successfully applied to the detection of antigen in malaria-infected human blood cultures at a 3% parasitemia level, and is assumed to detect parasite densities as low as 1,000 parasites.μL(-1).     

Production of porous silica microparticles by membrane emulsification

A method for the production of near-monodispersed spherical silica particles with controllable porosity based on the formation of uniform emulsion droplets using membrane emulsification is described. A hydrophobic metal membrane with a 15 μm pore size and 200 μm pore spacing was used to produce near-monodispersed droplets, with a mean size that could be controlled between 65 and 240 μm containing acidified sodium silicate solution (with 4 and 6 wt % SiO(2)) in kerosene. After drying and shrinking, the final silica particles had a mean size in the range between 30 and 70 μm. The coefficient of variation for both the droplets and the particles did not exceed 35%. The most uniform particles had a mean diameter of 40 μm and coefficient of variation of 17%. By altering the pH of the sodium silicate solution and aging the gel particles in water or acetone, the internal structure of the silica particles was successfully modified, and both micro- and mesoporous near-monodispersed spherical particles were produced with an average internal pore size between 1 and 6 nm and an average surface area between 360 and 750 m(2) g(-1). A material balance and particle size analysis provided identical values for the internal voidage of the particles, when compared to the voidage as determined by BET analysis.     

硫鸟嘌呤修饰的Mn:ZnS QDs磷光探针检测铂(Ⅲ)

通过水相合成法制备了硫鸟嘌呤(TG)修饰的锰掺杂硫化锌量子点(TG-Mn:ZnS QDs)。加入Pt⁴⁺后,Pt⁴⁺会与硫鸟嘌呤上的氮原子结合形成N-Pt⁴⁺配位结构附着在TG-Mn:ZnS QDs的表面,随着Pt⁴⁺浓度的增加,TG-Mn:ZnS QDs-Pt⁴⁺体系发生电子转移而导致磷光逐渐被猝灭,基于此构建了检测Pt⁴⁺的磷光探针。实验中考察了p H、时间对Pt⁴⁺猝灭TG-Mn:ZnS QDs磷光强度的影响。在最佳实验条件下,Pt⁴⁺浓度在0.06~2.4μg/mL范围内与TG-Mn:ZnS QDs的磷光强度呈良好的线性关系y=0.0884x+0.2319,R²=0.991,方法检出限(3σ/n)为1.3μg/mL。该磷光探针适用于实际样品中Pt⁴⁺含量的测定。     

Control of liquid crystal droplet configuration in polymer dispersed liquid crystal with macro-iniferter polystyrene

A polystyrene macro-iniferter was applied to control the alignment of liquid crystal molecules at the droplet wall of polymer dispersed liquid crystal (PDLC) films. The aspects of the alignment were monitored by observing the droplet in the PDLC film. With increasing the macro-iniferter polystyrene in the composition, the configuration of LC droplets changes from bipolar to radial. This is because the high concentration of the macro-iniferter polystyrene results in a small surface interaction between the LC and the polymer matrix, which favours the formation of radial configuration. The radial configuration was stable under our conditions. However, increasing the LC and the initiator concentrations resulted in the change from radial to bipolar.     

A quantified ginseng (Panax ginseng C.A. Meyer) extract influences lipid acquisition and increases adiponectin expression in 3T3-L1 cells

A Panax ginseng extract (PGE) with a quantified amount of ginsenosides was utilized to investigate its potential to inhibit proliferation, influence lipid acquisition and adiponectin expression in 3T3-L1 cells. Seven fingerprint ginsenosides were quantified using high performance liquid chromatography and their respective molecular weights were further confirmed via LC-ESI-MS analysis from four different extraction methods. Extraction using methanol under reflux produced significantly higher amounts of ginsenosides. The methanol extract consisted of Rg1 (47.40 ± 4.28 mg/g, dry weight of extract), Re (61.62 ± 5.10 mg/g), Rf (6.14 ± 0.28 mg/g), Rb1 (21.73 ± 1.29 mg/g), Rc (78.79 ± 4.15 mg/g), Rb2 (56.80 ± 3.79 mg/g), Rd (5.90 ± 0.41 mg/g). MTT analysis showed that PGE had a concentration-dependent cytotoxic effect on 3T3-L1 preadipocyte and the LC(50) value was calculated to be 18.2 ± 5 μg/mL. Cell cycle analysis showed minimal changes in all four phases. Differentiating adipocytes treated with ginseng extract had a visible decrease in lipid droplets formation measured by Oil red O staining. Consequently, triglycerides levels in media significantly (P < 0.05) decreased by 39.5% and 46.1% when treated at concentrations of 1 μg/mL and 10 μg/mL compared to untreated control cells. Western blot analysis showed that the adiponectin protein expression was significantly (P < 0.05) increased at 10 μg/mL, but not at 1 μg/mL. A quantified PGE reduced the growth of 3T3-L1 cells, down-regulated lipid accumulation and up-regulated adiponectin expression in the 3T3-L1 adipocyte cell model.     

Synthesis of rhodanine-bonded silica gel and its application in the preconcentration and separation of noble metals

A silica gel based sorbent containing rhodanine as functional group (RDSG) was prepared. Its adsorption and separation characteristics for Ag(I), Au(III) and Pd(II) were studied by flow-injection (FI) on-line preconcentration. Influence of different experimental parameters such as acidity, eluent, co-existing ions were investigated. Trace amounts of Ag, Au and Pd could be efficiently adsorbed by rhodanine-bonded silica gel from acidic solution and eluted with thiourea solution. Common co-existing ions exhibited virtually no interference to the preconcentration and determination. The adsorption capacity of RDSG was 0.0352, 0.107 and 0.122 mmol/g for Ag, Au and Pd, respectively. Detection limits of 0.004, 0.022 and 0.019 μg/mL for Ag, Au and Pd, respectively, were achieved with a sampling time of 60 s at a flow rate of 5.0 mL/min. The relative standard deviation were 0.5%, 0.9% and 1.7% for 0.040 μg/mL Ag, 0.200 μg/mL Au and 0.300 μg/¶mL Pd. The sorption property did not change after 1000 cycles of sorption-desorption. The contents of Ag and Au in three national certified ore samples and Pd in a secondary nickel alloy, an anode slime and a CoCl₂ electrolytic solution were determined. The results showed good agreement with the certified values.     

Fused silica capillaries with two segments of different internal diameters and inner surface roughnesses prepared by etching with supercritical water and used for volume coupling electrophoresis

In this work, single‐piece fused silica capillaries with two different internal diameter segments featuring different inner surface roughness were prepared by new etching technology with supercritical water and used for volume coupling electrophoresis. The concept of separation and online pre‐concentration of analytes in high conductivity matrix is based on the online large‐volume sample pre‐concentration by the combination of transient isotachophoretic stacking and sweeping of charged proteins in micellar electrokinetic chromatography using non‐ionogenic surfactant. The modified surface roughness step helped to the significant narrowing of the zones of examined analytes. The sweeping and separating steps were accomplished simultaneously by the use of phosphate buffer (pH 7) containing ethanol, non‐ionogenic surfactant Brij 35, and polyethylene glycol (PEG 10000) after sample injection. Sample solution of a large volume (maximum 3.7 μL) dissolved in physiological saline solution was injected into the wider end of capillary with inlet inner diameter from 150, 185 or 218 μm. The calibration plots were linear (R ² ∼ 0.9993) over a 0.060–1 μg/mL range for the proteins used, albumin and cytochrome c. The peak area RSDs from at least 20 independent measuremens were below 3.2%. This online pre‐concentration technique produced a more than 196‐fold increase in sensitivity, and it can be applied for detection of, e.g . the presence of albumin in urine (0.060 μg/mL).     

Dispersive Liquid‐Liquid Microextraction of Cu(II) Using a Novel Dioxime for Its Highly Sensitive Determination by Graphite Furnace Atomic Absorption Spectrometry

A dispersive liquid‐liquid microextraction (DLLME) technique was proposed for the enrichment and graphite furnace atomic absorption spectrometric (GFAAS) determination of Cu²⁺ in water samples. In this method a mixture of 480 μL acetone (disperser solvent) containing 26 μg S,S‐bis(2‐aminobenzyl)‐dithioglyoxime (BAT) ligand and 20 μL carbon tetrachloride (extraction solvent) was rapidly injected by a syringe into 5 mL aqueous sample containing copper ions (analyte). Thereby, a cloudy solution formed. After centrifugation, the fine droplets containing the extracted copper complex were sedimented at the bottom of the conical test tube. This phase was collected by a microsyring and after dilution by methanol, 20 μL of it was injected into the graphite tube of the instrument for analysis. Effects of some parameters on the extraction, such as extraction and disperser solvent type and volume, extraction time, salt concentration, pH and concentration of the chelating agent were optimized. The response surface method was used for optimization of the effective parameters on the extraction recovery. Under these conditions, an enrichment factor of 312 was obtained. The calibration graph was linear in the rage of 2–50 μ L⁻¹ Cu²⁺ with a detection limit of 0.03 μg L⁻¹ and a relative standard deviation (RSD) for five replicate measurements of 3.4% at 20 μg L⁻¹ Cu²⁺. The method was successfully applied to the determination of Cu²⁺ in some spring water samples.     

Bipolar to toroidal configuration transition in liquid crystal droplets

Liquid crystal (LC) droplets dispersed in isotropic media are of significant interest for scientific community and great importance for industries. The confinement can generate many fascinating LC director configurations and enable important practical applications. With tangential anchoring condition at the droplet surface, theoretically there are several possible configurations: bipolar, twisted bipolar, escaped toroidal, toroidal and so on. Bipolar configuration is usually observed in droplets made from common LCs while the toroidal configuration is rarely observed and it is hard to create especially in thermotropic LCs. Their realisations depend on the splay, bend and twist elastic constants ratio, and anchoring condition of the LC and polymer interface. We constructed thermotropic LCs with abnormally small bend elastic constants, with which stable toroidal configuration were successfully created. We provide a brand new method to create toroidal droplet by simply varying the bend elastic constant. We observed the transition from bipolar configuration to toroidal configuration. We performed a detailed study of the texture of toroidal droplets.     

Droplet size based separation by deterministic lateral displacement-separating droplets by cell--induced shrinking

We present a novel method for passive separation of microfluidic droplets by size at high throughput using deterministic lateral displacement (DLD). We also show that droplets containing Saccharomyces cerevisiae shrink significantly during incubation while droplets containing only yeast media retain or slightly increase their size. We demonstrate the DLD device by sorting out shrunken yeast-cell containing droplets from 31% larger diameter droplets which were generated at the same time containing only media, present at a >40-fold excess. This demonstrates the resolving power of droplet separation by DLD and establishes that droplets can be separated for a biological property of the droplet contents discriminated by a change of the physical properties of the droplet. Thus suggesting that this technique may be used for e.g. clonal selection. The same device also separates 11 μm from 30 μm droplets at a rate of 12,000 droplets per second, more than twofold faster than previously demonstrated passive hydrodynamic separation devices.     

Nematocidal flavone-C-glycosides against the root-knot nematode (Meloidogyne incognita) from Arisaema erubescens tubers

A screening of several Chinese medicinal herbs for nematicidal properties showed that Arisaema erubescens (Wall.) Schott tubers possessed significant nematicidal activity against the root-knot nematode (Meloidogyne incognita). From the ethanol extract, two nematicidal flavone-C-glycosides were isolated by bioassay-guided fractionation. The compounds were identified as schaftoside and isoschaftoside on the basis of their phytochemical and spectral data. Schaftoside and isoschaftoside possessed strong nematicidal activity against M. incognita (LC(50) = 114.66 μg/mL and 323.09 μg/mL, respectively) while the crude extract of A. erubescens exhibited nematicidal activity against the root-knot nematode with a LC(50) value of 258.11 μg/mL.     

Thermotropic liquid crystals embedded in a high water gel system

An emulsion was formed when the thermotropic liquid crystal (LC) mixture E5 was added to an aqueous polyvinylalcohol (pva) solution and shaken. This emulsion was gelled by addition of an aqueous borax solution. The pva polymer functioned not only as the gelling agent but also appeared to act as a polymeric surfactant which stabilised the LC droplets. This high water gel-liquid crystal (HWG-LC) system contained nearly 80 wt % water and more LC wt % than polymer. The system was thermally reversible, undergoing a gel to sol transition upon heating to 70°C and reforming a gel upon cooling. The HWG-LC showed electrooptical behaviour dependent upon a switched electric field when constrained between transparent electrodes. The pressure required to form a thin film between these electrodes induced a structural emulsion in the dispersion causing LC droplet disruption and the formation of an LC network in the gel.     

超支化聚合物作为放大信号的纳米磁性微球电化学免疫分析法

提出了一种基于超支化聚合物(HBP)固化酶标二抗作为放大信号和纳米磁球相结合的超灵敏的免疫分析新方法。首先羧基纳米磁性微球共价键合乙肝抗体(HBsAb)形成免疫磁性微球,然后与待测乙肝表面抗原(HBsAg)发生特异性结合,加入HBP标记的酶标二抗(HBPS)与结合的抗原结合发生夹心反应。在外加磁场的作用下,抗体抗原免疫复合物易从样品溶液中分离,在含有邻氨基苯酚和H2O2的底液中,快速生成具有电活性的化合物3-氨基吩呃嗪,用示差脉冲伏安法(DPV)测定响应电流,电流强度(I)与乙肝表面抗原浓度(c)在0.05～10.0μg/L范围内呈线性关系,线性回归方程为I(μA)=0.140+16.80 c(μg/L),相关系数r=0.9995,检出限达0.008μg/L,并用于实际样品的测定。     

磺胺嘧啶钠探针分析生物样品中蛋白质含量的研究

在pH 7.4的Tris-HCl缓冲溶液中,荧光猝灭光谱和三维荧光光谱显示磺胺嘧啶钠(SDS)可与人血清白蛋白(HSA)相互作用,使人血清白蛋白疏水微环境极性以及构象发生变化。考察了Δλ值、反应介质、离子强度等因素对该体系同步荧光光谱特征及强度的影响。在选定的最佳实验条件下,体系的同步荧光强度(ISF)与人血清白蛋白在1.38～276μg/mL的浓度范围内呈现良好的线性关系,线性相关系数为0.9992,从而建立了以磺胺嘧啶钠为分子探针,用固定波长同步荧光光谱分析测定蛋白质的新方法,检测限可达0.48μg/mL。用此方法对生物样品人血清、唾液和尿液中蛋白质含量进行了测定并进行加标回收实验,回收率在95.7%～103.7%之间。本文还用经典的考马斯亮蓝法对样品进行了测定,所得结果和本方法一致。同时还考察了一些常见的共存物质对蛋白质测定的影响。     

A new eudesmane sesquiterpene glucoside from Liriope muscari fibrous roots

The screening of several Chinese medicinal herbs for nematocidal properties showed that the ethanol extract of Liriope muscari fibrous roots possessed significant nematocidal activity against the pine wood nematode (Bursaphelenchus xylophilus). From the ethanol extract, a new constituent (1,4-epoxy-cis-eudesm-6-O-β-D-glucopyranoside) and three known glycosides [1β,6α-dihydroxy-cis-eudesm-3-ene-6-O-β-D-glucopyranoside (liriopeoside A), 1β,6β-dihydroxy-cis-eudesm-3-ene-6-O-β-D-glucopyranoside, and 1α,6β-dihydroxy-5,10-bis-epi-eudesm-4(15)-ene-6-O-β D-glucopyranoside] were isolated by bioassay-guided fractionation. The structures were elucidated by 1D and 2D NMR and MS techniques. 1,4-Epoxy-cis-eudesm-6-O-β-D-glucopyranoside possessed moderate nemato-cidal activity against B. xylophilus with a LC(50 )value of 339.76 μg/mL, while liriopeoside A (LC(50) = 82.84 μg/mL) and 1β,6β-dihydroxy-cis-eudesm-3-ene-6-O-β-D-glucopyranoside (LC(50) = 153.39 μg/mL) also exhibited nematocidal activity against B. xylophilus. The crude extract of L. muscari fibrous roots exhibited nematocidal activity against the pine wood nematode with a LC(50) value of 182.56 μg/mL.