含噁嗪环硅烷偶联剂对石英纤维/含硅芳炔复合材料性能的影响

从石英纤维(QF)、含硅芳炔树脂(PSA)分子结构特点出发,设计并合成了一种含有噁嗪环和端炔的新型硅烷偶联剂(BCA),并以BCA改善QF/PSA复合材料的界面性能。采用差示扫描量热分析(DSC)、红外光谱分析(FT-IR)、X-射线电子能谱(XPS)及扫描电镜(SEM)等测试手段表征了BCA与QF/PSA复合材料的相互作用和界面改性效果。结果表明:BCA能够分别与PSA和QF形成良好的化学键合,改善复合材料的界面黏结;经w=2.0%的BCA处理后,QF/PSA复合材料的层间剪切强度、弯曲强度分别较未处理前提高了69.1%和68.8%。     

介孔分子筛SBA-15的脂肪酶固定量分析测定

利用吸附法将假丝酵母脂肪酶(candida rugosa lipase,CRL)固定在介孔分子筛SBA-15上,对比了由单波长紫外分光光度法、双波长紫外分光光度法和二辛可宁酸法(bicinchoninic acid method, BCA)法测定的酶蛋白浓度及酶蛋白固定量.结果表明: SBA-15对紫外吸收有明显干扰,单波长紫外法测定结果远大于双波长紫外法和BCA法,双波长紫外法和BCA法测定结果较接近.利用BCA法测定了不同浓度CRL在介孔分子筛上的固定量,考察了固定化酶的泄漏量.在编号分别为Lu001和LLSD1的介孔分子筛SBA-15上的载酶量分别为16.6和114.12 mg/g.在缓冲溶液中SBA-15固定化酶的泄漏率只约为0.5%,可作为良好的酶固定化载体.     

纳米探针芯片技术用于微量乙肝病毒DNA的检测

利用两组探针修饰的微粒:(1)表面标记有可与待测乙肝病毒(HBV) DNA另一端结合的纳米金探针1(信号探针)以及可与信号探针部分结合的纳米金探针2(检测探针);(2)表面标记有可与待测HBV DNA一端结合的磁珠探针(捕捉探针1).检测靶HBV DNA时,磁珠探针与信号探针在液相中可分别与HBV DNA靶序列一端结合最终形成三明治样结构.再以磁场将三明治样复合物从反应液中分离,以DTT溶液将信号探针从纳米金颗粒上洗脱.洗脱后的信号探针数量反映靶基因的多寡,信号探针一段与预先点样的基因芯片上的捕捉探针2结合,检测探针与信号探针另一段相结合,最后用银染液将检测探针显色从而得到靶目标DNA相对定量信息.结果表明,本检测方法的检测灵敏度达到10-15 mol/L水平.检测时间少于1.5 h,检测结果与HBV DNA水平呈现较好的线性关系且无假阳性结果;本方法有望用于乙肝病人血清中HBV DNA的快速筛测及其它微生物基因的检测.     

A new method for non-labeling attomolar detection of diseases based on an individual gold nanorod immunosensor

Herein, we present the use of a single gold nanorod sensor for detection of diseases on an antibody-functionalized surface, based on antibody-antigen interaction and the localized surface plasmon resonance (LSPR) λ(max) shifts of the resonant Rayleigh light scattering spectra. By replacing the cetyltrimethylammonium bromide (CTAB), a tightly packed self-assembled monolayer of HS(CH(2))(11)(OCH(2)CH(2))(6)OCH(2)COOH(OEG(6)) has been successfully formed on the gold nanorod surface prior to the LSPR sensing, leading to the successful fabrication of individual gold nanorod immunosensors. Using prostate specific antigen (PSA) as a protein biomarker, the lowest concentration experimentally detected was as low as 111 aM, corresponding to a 2.79 nm LSPR λ(max) shift. These results indicate that the detection platform is very sensitive and outperforms detection limits of commercial tests for PSA so far. Correlatively, its detection limit can be equally compared to the assays based on DNA biobarcodes. This study shows that a gold nanorod has been used as a single nanobiosensor to detect antigens for the first time; and the detection method based on the resonant Rayleigh scattering spectrum of individual gold nanorods enables a simple, label-free detection with ultrahigh sensitivity.     

Broussochalcone A Is a Novel Inhibitor of the Orphan Nuclear Receptor NR4A1 and Induces Apoptosis in Pancreatic Cancer Cells

The orphan nuclear receptor 4A1 (NR4A1) is overexpressed in pancreatic cancer and exhibits pro-oncogenic activity, and NR4A1 silencing and treatment with its inactivators has been shown to inhibit pancreatic cancer cells and tumor growth. In this study, we identified broussochalcone A (BCA) as a new NR4A1 inhibitor and demonstrated that BCA inhibits cell growth partly by inducing NR4A1-mediated apoptotic pathways in human pancreatic cancer cells. BCA downregulated specificity protein 1 (Sp1)-mediated expression of an anti-apoptotic protein, survivin, and activated the endoplasmic reticulum (ER) stress-mediated apoptotic pathway. These results suggest that NR4A1 inactivation contributes to the anticancer effects of BCA, and that BCA represents a potential anticancer agent targeting NR4A1 that is overexpressed in many types of human cancers.     

Fabricating protein immunoassay arrays on nitrocellulose using dip-pen lithography techniques

Advancements in lithography methods for printing biomolecules on surfaces are proving to be potentially beneficial for disease screening and biological research. Dip-pen nanolithography (DPN) is a versatile micro and nanofabrication technique that has the ability to produce functional biomolecule arrays. The greatest advantage, with respect to the printing mechanism, is that DPN adheres to the sensitive mild conditions required for biomolecules such as proteins. We have developed an optimised, high-throughput printing technique for fabricating protein arrays using DPN. This study highlights the fabrication of a prostate specific antigen (PSA) immunoassay detectable by fluorescence. Spot sizes are typically no larger than 8 μm in diameter and limits of detection for PSA are comparable with a commercially available ELISA kit. Furthermore, atomic force microscopy (AFM) analysis of the array surface gives great insight into how the nitrocellulose substrate functions to retain protein integrity. This is the first report of protein arrays being printed on nitrocellulose using the DPN technique and the smallest feature size yet to be achieved on this type of surface. This method offers a significant advance in the ability to produce dense protein arrays on nitrocellulose which are suitable for disease screening using standard fluorescence detection.     

Multiplex immunoassays on size-categorized individual beads using time-resolved fluorescence

Miniaturized multiplex immunoassays were studied on individual beads to detect analytes in the same reaction mixture. With this approach hands-on time, cost, and amount of reagents as well as waste produced in the assays were reduced. Particles were categorized according to size for immunoassays of prostate specific antigen (PSA) and acute myocardial infarction (AMI) markers. Additionally, free and dual PSA assays were carried out on a single bead. The analyte concentration was detected directly on the surface of the beads using stable, intrinsically fluorescent europium and terbium chelates, and time-resolved fluorometry. Less than 0.4 ng ml⁻¹ PSA (corresponding to ca. 10 amol) was detected in free, dual and multiplex PSA assays and 0.1 and 2.4 ng ml⁻¹ of myoglobin (Mb) and creatine kinase MB (CK-MB), respectively, were monitored in the multiplex AMI marker assay. The effect of bead size and material on free PSA detection was also investigated. The beads were detected three times under different conditions; in liquid, after drying and after dissociating europium ions from the chelate into the DELFIA^® fluorescence enhancement solution. The same detection sensitivity was found for the dissociative and non-dissociative methods indicating that the current labeling technology for the surface detection is comparable to the commercial DELFIA system.     

A facile,one‐step method for the determination of accessible surface primary amino groups on solid carriers

Amino‐functionalized microspheres are emerging as promising candidates for biomedical applications. Quantification of surface amino groups is of great significance to achieve a rational surface design and to facilitate subsequent bioconjugation. In this paper, we describe a facile method [named as subtractive 2‐iminothiolane (ITL)/bicinchnoninic acid (BCA) method] to determine the accessible surface primary amino groups using BCA and ITL on the basis of the rapid color reaction between ITL and BCA at ambient temperature. ITL was used to quantitatively label the surface amino groups, and the ITL consumed by the amino groups was determined by BCA in a solution reaction. The amino density of amino‐functionalized silica microspheres, polystyrene microspheres and magnetic microspheres were determined. The present method we proposed exhibits great simplicity (one‐step labeling and immediate detection without washing procedure) with high precision (standard error <5% for each concentration point of standards) and specificity. Copyright © 2012 John Wiley & Sons, Ltd.     

Interference study should be performed for every protein measurement method used

It is particularly important, when analyzing biological material, for the measurement procedure to be specific to the analyte and not to suffer interference by the matrix effect. Tissue fraction studies also require rapid and accurate methods to estimate the concentration of protein in solutions as well as many measurement methods used in medical laboratories. The design of this study is based on a comparison of the Lowry and the bicinchoninic acid (BCA) methods for the measurement of the total protein concentrations of rat liver subcellular fractions. In our experiment, subcellular fractions enriched in peroxisomes (POs) obtained by differential centrifugation were then further separated by means of density gradient centrifugation. We performed the protein measurement assays on all fractions obtained during the purification steps. The protein contents of the fractions obtained were determined by the two methods. The method comparison statistics were performed by linear Deming regression analysis and Altman and Bland bias plot. The regression equation was unacceptable, indicating that the last three fractions separated by means of Nycodenz discontinuous density gradient centrifugation gave remarkably divergent results. For the Lowry method, the Nycodenz effect could not be eliminated with the use of interference blank. In addition to Nycodenz, the potentially interfering compound used in the isolation procedure as isolation medium was 3-(morpholino)propane sulfonic acid (MOPS). In decreased concentrations of MOPS (10 mM), interference blank should be used for correct measurement with Lowry, but in practical use 10 mM does not provide buffering potency. In the BCA method, interference blank correction seemed to eliminate the measurement error in all concentrations of Nycodenz. There was no MOPS effect on the BCA measurement assay. Referring to deviations as sample-inherent matrix effects, we concluded that not only one, but more measurement methods should be used in order to make a correct protein measurement.     

Carbon nanotube amplification strategies for highly sensitive immunodetection of cancer biomarkers

We describe herein the combination of electrochemical immunosensors using single-wall carbon nanotube (SWNT) forest platforms with multi-label secondary antibody-nanotube bioconjugates for highly sensitive detection of a cancer biomarker in serum and tissue lysates. Greatly amplified sensitivity was attained by using bioconjugates featuring horseradish peroxidase (HRP) labels and secondary antibodies (Ab(2)) linked to carbon nanotubes (CNT) at high HRP/Ab(2) ratio. This approach provided a detection limit of 4 pg mL(-)(1) (100 amol mL(-)(1)), for prostate specific antigen (PSA) in 10 microL of undiluted calf serum, a mass detection limit of 40 fg. Accurate detection of PSA in human serum samples was demonstrated by comparison to standard ELISA assays. PSA was quantitatively measured in prostate tissue samples for which PSA could not be differentiated by the gold standard immunohistochemical staining method. These easily fabricated SWNT immunosensors show excellent promise for clinical screening of cancer biomarkers and point-of-care diagnostics.     

A novel electrochemical immunosensor based on colabeled silica nanoparticles for determination of total prostate specific antigen in human serum

A novel electrochemical immunosensor using functionalized silica nanoparticles (Si NPs) as protein tracer has been developed for the detection of prostate specific antigen (PSA) in human serum. The immunosensor was carried out based on a heterogeneous sandwich procedure. The PSA capture antibody was immobilized on the gold electrode via glutaraldehyde crosslink. After reaction with the antigen in human serum, Si NPs colabeled with detection antibody and alkaline phosphatase (ALP) was sandwiched to form the immunocomplex on the gold electrode. ALP carried by Si NPs convert nonelectroactive substrate into the reducing agent and the latter, in turn, reduce metal ions to form electroactive metallic product on the electrode. Linear sweep voltammetry (LSV) was used to quantify the amount of the deposited silver and give the analytical signal for PSA. The parameters including the concentration of the ALP used to functionalize the Si NPs and the enzyme catalytic reaction time have been studied in detail and optimized. Under the optimum conditions of immunoreaction and electrochemical detection, the electrochemical immunosensor was able to realize a reliable determination of PSA in the range of 1–35 ng/mL with a detection limit of 0.76 ng/mL. For six human serum samples, the results performed with the electrochemical immunosensor were in good agreement with those obtained by chemiluminescent microparticle immunoassay (CMIA), indicating that the electrochemical immunosensor could satisfy the need of practical sample detection.     

Denaturation of proteins by SDS and tetraalkylammonium dodecyl sulfates

This article describes the use of capillary electrophoresis (CE) to examine the influence of different cations (C(+); C(+) = Na(+) and tetra-n-alkylammonium, NR(4)(+), where R = Me, Et, Pr, and Bu) on the rates of denaturation of bovine carbonic anhydrase II (BCA) in the presence of anionic surfactant dodecylsulfate (DS(-)). An analysis of the denaturation of BCA in solutions of Na(+)DS(-) and NR(4)(+)DS(-) (in Tris-Gly buffer) indicated that the rates of formation of complexes of denatured BCA with DS(-) (BCA(D)-DS(-)(n,sat)) are indistinguishable and independent of the cation below the critical micellar concentration (cmc) and independent of the total concentration of DS(-) above the cmc. At concentrations of C(+)DS(-) above the cmc, BCA denatured at rates that depended on the cation; the rates decreased by a factor >10(4) in the order of Na(+) ≈ NMe(4)(+) > NEt(4)(+) > NPr(4)(+) > NBu(4)(+), which is the same order as the values of the cmc (which decrease from 4.0 mM for Na(+)DS(-) to 0.9 mM for NBu(4)(+)DS(-) in Tris-Gly buffer). The relationship between the cmc values and the rates of formation of BCA(D)-DS(-)(n,sat()) suggested that the kinetics of denaturation of BCA involve the association of this protein with monomeric DS(-) rather than with micelles of (C(+)DS(-))(n). A less-detailed survey of seven other proteins (α-lactalbumin, β-lactoglobulin A, β-lactoglobulin B, carboxypeptidase B, creatine phosphokinase, myoglobin, and ubiquitin) showed that the difference between Na(+)DS(-) and NR(4)(+)DS(-) observed with BCA was not general. Instead, the influence of NR(4)(+) on the association of DS(-) with these proteins depended on the protein. The selection of the cation contributed to the properties (including the composition, electrophoretic mobility, and partitioning behavior in aqueous two-phase systems) of aggregates of denatured protein and DS(-). These results suggest that the variation in the behavior of NR(4)(+)DS(-) with changes in R may be exploited in methods used to analyze and separate mixtures of proteins.     

Eliminating positively charged lysine epsilon-NH3+ groups on the surface of carbonic anhydrase has no significant influence on its folding from sodium dodecyl sulfate

This study compares the folding of two polypeptides--bovine carbonic anhydrase (BCA) and peracetylated BCA (BCA-Ac(18))--having the same sequence of amino acids but differing by 18 formal units of charge, from a solution containing denaturing concentrations of sodium dodecyl sulfate (SDS). Acetylation of BCA with acetic anhydride converts all 18 lysine-epsilon-NH(3)(+) groups to lysine-epsilon-NHCOCH(3) groups and generates BCA-Ac(18). Both BCA and BCA-Ac(18) are catalytically active, and circular dichroism spectroscopy (CD) suggests that they have similar secondary and tertiary structures. SDS at concentrations above approximately 10 mM denatured both proteins. When the SDS was removed by dialysis, both proteins were regenerated in native form. This study suggests that large differences in the net charge of the polypeptide have no significant influence on the structure, the ability to refold, or the rate of refolding of this protein from solutions containing SDS. This study reinforces the idea that charged residues on the surface of BCA do not guide protein folding and raises the broader question of why proteins have charged residues on their surface, outside of the region of the active site.     

Enantiomeric separation of basic drugs with partially filled serum albumin as chiral selector in capillary electrophoresis.

A reliable method is presented for the chiral separation of three basic drugs (mexiletine, chlorpheniramine and propranolol) with serum albumins (human and porcine, HSA and PSA) as chiral selectors by capillary electrophoresis in combination with the partial filling technique. Based on the systematic optimization of operation variables, the chiral separation of mexiletine, chlorpheniramine and propranolol was achieved in the pH 7.4 phosphate buffer by using HSA, PSA and PSA as selectors, respectively. The chiral recognition ability of HSA and PSA was compared. HSA and PSA show a different chiral recognition ability for each of the three drugs. In addition, the association constants between enantiomeric drugs and proteins were determined to be 2.00 and 3.80 x 10(2) M(-1) for mexiletine and HSA, 0.59 and 1.12 x 10(3) M(-1) for chlorpheniramine and PSA, and 0.87 and 1.42 x 10(3) M(-1) for propranolol and PSA. The method for the chiral separation and determination of association constants possesses the advantages of simple performance, effective avoiding of the interference of the UV detection from protein, and lowering of the reagent consumption.     

一种基于二喹啉甲酸- Cu+显色反应的毛细管电泳检测蛋白质的新方法

采用毛细管电泳法和蛋白质显色反应-二喹啉甲酸(BCA)法,结合微波辅助反应,在60 mmol/L硼酸盐缓冲液(pH 9.5)中,实现了对蛋白质的快速毛细管电泳分析检测。同时以β-环糊精为包合添加剂,实现了BCA-Cu+复合物和游离BCA的分离,从而在波长200 nm处以测定特征生成的BCA-Cu+复合物来间接检测蛋白质,其峰强度比直接检测蛋白质自身吸收的峰强度提高了2个数量级。对于转铁蛋白、蓖麻毒素,其线性范围分别为2～200 mg/L和2～100 mg/L,检出限分别为0.33和0.37 mg/L。将该方法成功地应用于第一届蓖麻毒素国际实验室间比对测试的部分样品,含量测定结果满意。     

Photoelectrochemical immunoassay for microcystin-LR based on a fluorine-doped tin oxide glass electrode modified with a CdS-graphene composite

We report on a sensitive electrochemical aptasensor for the detection of human prostate specific antigen (PSA). It is based on the signal amplification of the biotin-avidin system using a sensing platform that is making use of a graphite electrode modified with gold nanoparticles that were covered with graphitized mesoporous carbon nanoparticles (AuNPs@GMCs). The AuNPs@GMCs hybrid was prepared by linking 1,6-hexanedithiol-functionalized GMCs and gold nanoparticles via Au-S groups. Then, streptavidin was immobilized on the electrode modified with the AuNPs@GMCs so to enlarge the amount of biotin-aptamer which led to enhanced detection sensitivity. If an PSA aptamer captures the target PSA on the electrode, the differential pulse voltammetric (DPV) signal of the hexacyanoferrate redox system decreases. Factors affecting the performance of the aptasensor were studied in detail. Under optimal conditions, the DPV signal changes could be used to quantitatively detect PSA in the concentration range from 0.25 to 200?ng?mL^?1, with a lowest limit of detection as small as 0.25?ng?mL^?1. The aptasensor is highly specific and displays acceptable precision, good stability and repeatability.     

Prostate-specific antigen immunoassays on the BioCD

The BioCD is a spinning-disc interferometric biosensor on which antibodies are immobilized to capture target antigens from biological samples. In this work, BioCDs measured the interferometric response to prostate-specific antigen (PSA). The ideal detection limit for PSA was determined using a BioCD with 12,500 printed target antibody spots with a corresponding number of reference protein spots. Statistical analysis projects the detection limit of PSA as a function of the number of spots included in the average. When approximately 10,000 spot pairs were averaged, the 3σ detection limit was 60 pg/ml in a 2 mg/ml simple protein background. A standard format for BioCD immunoassays uses 96 wells with 32 target spots paired with reference spots. In serum, the detection limit for this format was 1 ng/ml in 3:1 diluted female human serum using a sandwich assay with a nonfluorescent mass tag.     

Novel solution-phase immunoassays for molecular analysis of tumor markers

at 3 x 10(9) M(-1) and a step-wise binding process with PSA-free MAB. Thus, this solution-phase quantitative ECL immunoassay allowed measurement of the affinity of serum PSAs with their MABs and screening of PSAs based upon their affinity to MABs. Unlike other immunoassays, this immunoassay demonstrated one-step rapid analysis while simultaneously eliminating immobilization, separation and washing steps and detected PSA at a level of 1.7 pg mL(-1), which is 1000-fold more sensitive than current PSA immunoassays. Furthermore, single-molecule (SM) phosphorescence microscopy was developed to detect single serum PSA-free and PSA-complex molecules in solution with no use of antibody showing that PSA-free molecules diffused faster than PSA-complex molecules in solution. This finding is consistent with ECL measurements and implies the possibility of screening individual analytes in a complex mixture using their distinct SM diffusion distance. This is the first report describing the detection of single protein molecules labeled with a metal-complex using phosphorescence microscopy and also the screening of serum tumor markers using ECL and SM phosphorescence solution-phase assays.     

Electrochemical immunoassay for the prostate specific antigen using ceria mesoporous nanospheres

We report on a sensitive electrochemical immunoassay for the prostate specific antigen (PSA). An immunoelectrode was fabricated by coating a glassy carbon electrode with multiwalled carbon nanotubes, poly(dimethyldiallylammonium chloride), CeO₂ and PSA antibody (in this order) using the layer-by-layer method. The immunosensor is then placed in a sample solution containing PSA and o-phenylenediamine (OPD). It is found that the CeO₂ nanoparticles facilitate the electrochemical oxidation of OPD, and this produces a signal for electrochemical detection of PSA that depends on the concentration of PSA. There is a linear relationship between the decrease in current and the concentration of PSA in the 0.01 to 1,000 pg mL^?1 concentration range, and the detection limit is 4 fg mL^?1. The assay was successfully applied to the detection of PSA in serum samples. This new differential pulse voltammetric immunoassay is sensitive and acceptably precise, and the fabrication of the electrode is well reproducible. Figure

A novel electrochemical immunoassay for prostate specific antigen (PSA) was developed. Ceria (CeO₂) mesoporous nanospheres facilitated the electrochemical oxidation of o-phenylenediamine (OPD). The developed immunoassay has high sensitivity and can be successfully applied for the detection of PSA in serum samples     

On-chip graphene oxide aptasensor for multiple protein detection

The versatility of an on-chip graphene oxide (GO) aptasensor was successfully confirmed by the detection of three different proteins, namely, thrombin (TB), prostate specific antigen (PSA), and hemagglutinin (HA), simply by changing the aptamers but with the sensor composition remaining the same. The results indicate that both DNA and RNA aptamers immobilized on the GO surface are sufficiently active to realize an on-chip aptasensor. Molecular selectivity and concentration dependence were investigated in relation to TB and PSA detection by using a dual, triple, and quintuple microchannel configuration. The multiple target detection of TB and PSA on a single chip was also demonstrated by using a 2 × 3 linear-array GO aptasensor. This work enables us to apply this sensor to the development of a multicomponent analysis system for a wide variety of targets by choosing appropriate aptamers.