补体C1q压电免疫传感器的研究

研制了一种可重复使用的压电免疫传感器用于检测补体C1q.比较了聚乙烯亚胺粘附、戊二醛交联法,物理吸附法以及蛋白A法三种固定化抗体蛋白的方法.使用蛋白A法,补体C1q在所测浓度范围内有良好的线性关系.使用过的晶片用0.1 mol/L柠檬酸盐缓冲溶液(pH 3.0)解吸,可重复使用4次.     

Detection of antisperm antibody in human serum using a piezoelectric immunosensor based on mixed self-assembled monolayers

A novel piezoelectric immunosensor based on mixed self-assembled monolayers (mixed SAMs) formed by short-chain amine- and carboxyl-terminated thiols has been developed to immobilize antigens onto gold electrodes for detecting antisperm antibody (AsAb) in human serum samples. The properties and the enhanced performance of the affinity biosensor interface based on mixed SAMs are investigated. Most importantly, analytical results of several human serum samples using the developed technique are in satisfactory agreement with those given by the enzyme-linked immunosorbent assay (ELISA) method in the concentration ranging from 32.3 to 300.0 mU/ml. It means the procedure proposed in this paper is likely to have a great potential in research and may play an important clinical role in a few years later.     

Detection of complement C1-inhibitor with a piezoelectric immunosensor

A novel piezoelectric immunosensor has been developed for the detection of human complement C1-inhibitor. Anti-C1-inhibitor antibody was immobilized onto the gold electrodes of a 9 MHz AT-cut piezoelectric crystal. Coating the crystal with polyethyleneimine adhesion, followed by a glutaraldehyde cross-linking method to immobilize antibody showed better results than the physical adsorption method with respect to sensitivity and reproducibility. Under the optimized experimental conditions, the sensor showed good response to the C1-inhibitor in the range from 2.0 × 10^–8 to 1.2 × 10^–6 g. Other proteins in human serum did not remarkably interfere with the detection. The crystals could be regenerated 5 times, when bound materials on the crystal surface were eluted by strong acid and strong alkali solution and subsequently cleaned in an ultrasonic cleaner.     

Piezoelectric immunosensor based on magnetic nanoparticles with simple immobilization procedures

A novel method for immobilizing antibodies (antigens) based on magnetic nanoparticles has been proposed for piezoelectric immunoassay. The goat-anti-IgG antibody (IgGAb) as the model analyte was first covalently immobilized to magnetic nanoparticles, which were surface modified with amino-groups. The magnetic bio-nanoparticles (MBN-s) formed were attached to the surfaces of quartz crystal with the help of a permanent magnet. The detection of immunoglobulin G (IgG) was performed with the sensor prepared. The process of immobilization and immunoreaction was monitored by frequency recording. From the SEM images of the sensor surface before and after immobilization of MBN, one can see that the MBN was homogeneously adsorbed on sensor surface. The piezoelectric immunosensor can determine IgG in the range of 0.6-34.9 μg ml⁻¹ with a detection limit of 0.36 μg ml⁻¹. The MBN and immunocomplex layer can easily be removed simply by taking away the magnetic field, making the piezoelectric sensor easy to be regenerated.     

Detection of complement C1-inhibitor with a piezoelectric immunosensor

A novel piezoelectric immunosensor has been developed for the detection of human complement C1-inhibitor. Anti-C1-inhibitor antibody was immobilized onto the gold electrodes of a 9 MHz AT-cut piezoelectric crystal. Coating the crystal with polyethyleneimine adhesion, followed by a glutaraldehyde cross-linking method to immobilize antibody showed better results than the physical adsorption method with respect to sensitivity and reproducibility. Under the optimized experimental conditions, the sensor showed good response to the C1-inhibitor in the range from 2.0 x 10(-8) to 1.2 x 10(-6) g. Other proteins in human serum did not remarkably interfere with the detection. The crystals could be regenerated 5 times, when bound materials on the crystal surface were eluted by strong acid and strong alkali solution and subsequently cleaned in an ultrasonic cleaner.     

A piezoelectric immunosensor for the determination of pesticide residues and metabolites in fruit juices

A quartz crystal microbalance (QCM) immunosensor was developed for the determination of the insecticide carbaryl and 3,5,6-trichloro-2-pyridinol (TCP), the main metabolite of the insecticide chlorpyrifos and of the herbicide triclopyr. The detection was based on a competitive conjugate-immobilized immunoassay format using monoclonal antibodies (MAbs). Hapten conjugates were covalently immobilized, via thioctic acid self-assembled monolayer (SAM), onto the gold electrode sensitive surface of the quartz crystal. This covalent immobilization allowed the reusability of the modified electrode surface for at least one hundred and fifty assays without significant loss of sensitivity. The piezoimmunosensor showed detection limits (analyte concentrations producing 10% inhibition of the maximum signal) of 11 and 7 μg l⁻¹ for carbaryl and TCP, respectively. The sensitivity attained (I₅₀ value) was around 30 μg l⁻¹ for both compounds. Linear working ranges were 15-53 μg l⁻¹ for carbaryl and 13-83 μg l⁻¹ for TCP. Each complete assay cycle took 20 min. The good sensitivity, specificity, and reusability achieved, together with the short response time, allowed the application of this immunosensor to the determination of carbaryl and TCP in fruits and vegetables at European regulatory levels, with high precision and accuracy.     

蛋白A定向固定抗体的纤维蛋白压电免疫传感器的研究

将9MHz双面镀金石英晶体浸入蛋白A溶液中,在晶体电极表面形成一层均匀的蛋白A薄层,用于定向固定人体纤维蛋白抗体.在蛋白A层上形成一层有序致密的自组装抗体分子膜,研制成一种新型的用于人体纤维蛋白检测的压电免疫传感器.比较了3种固定抗体方法的效果,从传感器的灵敏度、稳定性、重现性等考虑,蛋白A吸附法优于聚乙烯亚胺及牛血清白蛋白固定抗体的方法.研究了蛋白A浓度、抗体效价以及抗原抗体反应时间等对传感器灵敏度的影响,考察了电极的选择性和再生能力.纤维蛋白在1×10^-4～1×10^-2g/L浓度范围内有良好响应.     

基于双醛基功能化离子液体构建电化学免疫传感器

合成了含双醛基的离子液体,此离子液体一端的醛基与修饰在电极表面的氨基发生共价键作用,将离子液体修饰在电极表面,另一端的醛基可用来固定抗体,构建电化学免疫传感器,实现对心肌肌钙蛋白I(cTnI)的检测。离子液体通过共价键作用固定在电极表面,不仅减少了从电极表面向检测溶液的渗透,提高传感器的稳定性,而且还可以直接固定抗体,不需要使用其他交联试剂;同时,离子液体可增强传感界面的导电性,提高传感器的灵敏度。在优化的实验条件下,传感器的线性范围为0.1~40 ng/mL,检出限为0.06 ng/mL。     

压电免疫传感器用于B因子的测定

B因子是一种不耐热的球蛋白,它参与机体的防御,在炎症过程、细胞和组织损伤中均起重要作用.B因子的检测方法常用的有单扩散法、火箭电泳法和溶血法,前者灵敏度不高,重复性差;后两者操作较复杂^[1,2].     

压电免疫传感器用于C2型葡萄球菌肠毒素的测定

报道了用蛋白Ａ－金电极法将抗体固化在３．５８ＭＨｚ和１０ＨｚＡＴ切割的石英晶体上，制作的可重复使用的压电免疫传感器，测定Ｃ２型葡萄球菌肠毒素。最适ＰＨ值为７，并用不同的方法测试了晶体片上的抗体固化情况。     

A novel piezoelectric immunosensor for detection of carcinoembryonic antigen

A simple, rapid, and highly sensitive immunosensor for the direct determination of carcinoembryonic antigen (CEA) in human serum using a piezoelectric crystal has been developed and optimized. In order to improve sensitivity of the immunosensor, a protein A-based orientation-controlled immobilization method for antibodies was adopted together with an immunoreactive accelerant of polyethyleneglycol (PEG) used to amplify the signal response of frequency. Human normal serum was utilized as a reference background. The linear range for CEA concentration obtained by the end-point method was 66.7-466.7 ng/mL. Clinical samples from cancer patients were analyzed by the proposed piezoelectric immunoassay, and the analytical results were reasonably comparable with those obtained by the chemiluminescence immunoassay (CLIA). The proposed immunosensor provides a new promising method for the highly sensitive immunoassay of CEA in clinical laboratory.     

基于功能化N掺杂C3N4构建光电免疫传感器检测癌胚抗原

分别合成掺杂不同比例N的C3N4纳米材料,利用红外光谱、荧光光谱等技术筛选出光电性能最优的N掺杂C3N4,并通过透射电镜、红外光谱等进行表征。将其作为光电转换材料,在氧化铟锡(ITO)电极表面构建高性能的免疫传感器。通过抗原-抗体的高特异性结合,实现对肿瘤标志物癌胚抗原的高灵敏检测。光电流结果表明:电流响应随癌胚抗原浓度的增加而减小,并在1.0 pg/mL^10 ng/mL的范围内呈现良好线性相关性。将此方法应用于检测血清样品中的癌胚抗原浓度,检测结果与商业化的酶联免疫分析方法结果进行对比没有显著差异。     

蛋白A与联硫基二(琥珀酰亚胺丙酸盐)修饰电化学免疫传感器测定雌二醇(英文)

将抗体结合蛋白A和交联剂联硫基二(琥珀酰亚胺丙酸盐)(DTSP)组成的骨架修饰到电极表面,再将单克隆雌二醇抗体与骨架中的蛋白A相结合,制备出电化学免疫传感器.利用样品雌二醇与辣根过氧化酶所标记的雌二醇同传感器表面抗体的竞争性结合,将该传感器用于测定溶液中样品雌二醇的浓度;利用方波伏安法监测电化学还原辣根过氧化酶催化产生的苯醌分子的电流,以还原电流为纵坐标,以雌二醇浓度为横坐标绘制标准曲线.结果表明,雌二醇浓度在50～1 500ng.L-1范围内与方波伏安还原电流呈良好的线性关系,灵敏度为0.51μA.ng-1.L,检测限为50ng.L-1.所制备的免疫传感器良好的分析性能得益于蛋白A和DTSP所组成的骨架,该骨架能够增加免疫分子组分在电极表面的修饰量,并能够控制抗体在电极上的结合方位,使其抗原结合位点朝向电极外端,减少结合空间阻碍.     

基于生物素-亲和素系统的压电免疫传感器检测相思子毒素研究

利用生物素-亲和素系统的放大作用和纳米金质量扩增效应,建立了压电免疫传感器检测相思子毒素的新方法.首先在石英晶体的金电极上依次组装二巯基丙酸、EDC和NHS进行表面修饰,然后通过亲和素固定生物素标记相思子毒素多抗来制备敏感膜,利用纳米金的质量扩增效应设计了一种"毒素-纳米金标记单抗"复合物,成功实现了对相思子毒素的检测,提高了传感器灵敏度和重现性.本传感器对相思子毒素响应的线性范围为0.05～5 mg/L; 回归方程为Δf＝50.81CAbrin+67.11(r=0.9903,n＝10,P<0.0001); 检测灵敏度为50.81 Hz · L/mg.     

Stabilization Studies on Divalent Five‐Membered Rings C4H4C,C4H4Si,C4H4Ge,C4H4Sn,and C4H4Pb

Energy differences, ΔX_S‐t (X = E, H and G) (ΔX_S‐t = X_(singlet)‐X_(triplet)) between singlet (s) and triplet (t) states are calculated at B3LYP/6‐311++G (3df,2p). The DFT calculations show that the triplet state of C₄H₄C is a ground state with planar conformer respect to its corresponding nonplanar singlet state. Both singlet and triplet states of C₄H₄M (M = Si, Ge, Sn and Pb) have a planar conformer with the singlet ground state. Four isodesmic reactions are presented for determining the stability energies, SE. NICS calculations are carried out for C₄H₄M to determine the aromatic character.     

Picogram-detection of estradiol at an electrochemical immunosensor with a gold nanoparticle|Protein G-(LC-SPDP)-scaffold

Low picograms of the hormone 17β-estradiol were detected at an electrochemical immunosensor. This immunosensor features a gold nanoparticle|Protein G-(LC-SPDP)¹-scaffold, to which a monoclonal anti-estradiol capture antibody was immobilised to facilitate a competitive immunoassay between sample 17β-estradiol and a horseradish peroxidase-labelled 17β-estradiol conjugate. Upon constructing this molecular architecture on a disposable gold electrode in a flow cell, amperometry was conducted to monitor the reduction current of benzoquinone produced from a catalytic reaction of horseradish peroxidase. This current was then quantitatively related to 17β-estradiol present in a sample. Calibration of immunosensors in blood serum samples spiked with 17β-estradiol yielded a linear response up to ∼1200 pg mL⁻¹, a sensitivity of 0.61 μA/pg mL⁻¹ and a detection limit of 6 pg mL⁻¹. We attribute these favourable characteristics of the immunosensors to the gold nanoparticle|Protein G-(LC-SPDP) scaffold, where the gold nanoparticles provided a large electrochemically active surface area that permits immobilisation of an enhanced quantity of all components of the molecular architecture, while the Protein G-(LC-SPDP) component aided in not only reducing steric hindrance when Protein G binds to the capture antibody, but also providing an orientation-controlled immobilisation of the capture antibody. Coupled with amperometric detection in a flow system, the immunosensor exhibited excellent reproducibility.     

Diagnosis of tularemia using piezoelectric biosensor technology

A piezoelectric immunosensor for indirect diagnosis of tularemic infection in mouse serum was developed. Francisella tularensis LVS antigen was covalently immobilized on the sensing surface using cystamine and glutaraldehyde for activation and modification of the gold electrode. The normal mouse serum (NMS) and serum prepared from mice immunized by Escherichia coli were used as negative controls providing signal of 28 Hz during a 5 min interaction. The tularemic infectious (immunized) mouse serum (IMS) as sample resulted in the signal above 75 Hz (fifth day after infection). The control sensor containing bovine serum albumin as sensing element provided a signal below 5 Hz with NMS as well IMS. The effects of dilution degree and purification of sera were tested. To improve resolution of the method, sample pretreatment steps such as precipitation with ammonium sulphate and immunoglobulin extraction on CBind™ L and MEP HyperCel columns were tested. R.S.D. of measurements was 2.3% for NMS and 2.4% for IMS, respectively. The developed method allows to indicate the presence of anti-tularemic antibodies shortly (1-3 days) after infection, one analysis is completed in 10 min.     

IgG免疫纳米探针凝聚反应压电传感检测

IgG是人血清中主要抗感染的抗体, 约占成人血清免疫球蛋白总量的75%, 被视为判断健康或疾病的一个指标. 本文通过对IgG的检测, 探讨了纳米探针免疫凝聚压电传感方法的可行性.     

Detection of β-thalassemia by a DNA piezoelectric biosensor coupled with polymerase chain reaction

β-Thalassemia is an inherited disorder mainly caused by mutations in the gene of the β-globin chain of adult haemoglobin (HbA). Clinically, β-thalassemia can be a mild or silent condition, or it can cause severe diseases, leading to transfusion dependence. Studies at the gene level have identified a large number of variations in the β-globin gene in different populations. In the Mediterranean area one of the most common mutation is the C → T substitution in the codon 39 of the gene.A new procedure for detecting codon 39 mutation in the β-globin gene is reported, based on a DNA piezoelectric biosensor. An oligonucletidic probe (25-mer), specific for the region around codon 39, is immobilised on the gold surface of a piezoelectric quartz crystal. The hybridisation between the immobilised probe and the complementary strand in solution is detected recording the variations of the crystal frequency.Experiments with synthetic oligonucleotides were initially performed. Distinguishable frequency shifts were obtained from the interaction between the immobilised probe and the complementary and the mismatch oligonucleotides. A solution containing 50% of both the oligonucleotides has been also tested and distinguished from the others evaluating the resulting signals. Experiments with non-complementary oligonucleotides gave no signal variation. The biosensor was able to distinguish between sequences differing in only one base also using polymerase chain reaction-amplified samples [771 base pairs (bp)] of DNA extracted from human blood of thalassemic and healthy (normal) patients or patients with β-thalassemia traits.The optimised DNA piezoelectric biosensor has been successfully applied to the determination of one of the most frequent mutation characteristic of β-thalassemia in the Mediterranean population.     

Development of a functional impedimetric immunosensor for accurate detection of thyroid-stimulating hormone

Thyroid-stimulating hormone (TSH), which regulates the synthesis of thyroid gland hormones affecting the whole metabolism, is a pituitary hormone. Determination of TSH is crucial for monitoring thyroid gland-related disorders and some metabolic diseases.In this study, a nonlabeled immunosensor based on covalent immobilization of anti-TSH antibody by using the formation of self-assembled monolayers (SAM) of 4-mercaptophenylacetic acid (4-MPA) and functionalization of carboxyl ends with 1-ethyl-3-(3-dimetilaminopropil) carbodiimide (EDC)/N-Hydroxysuccinimide (NHS) was fabricated for detection of TSH. Immobilization steps including the concentration of 4-MPA, the concentration of anti-TSH antibody, and duration of anti-TSH antibody incubation were optimized by utilizing electrochemical impedance spectroscopy. Under optimal conditions, a sensitive, rapid, and accurate determination of TSH at a concentration range between 0.7 and 3.5 mIU/L was accomplished with a notable linearity and LOD value of 0.034 mIU/L, as well as reproducibility and repeatability. Moreover, for comparison, linear range experiments were also carried out by using other electrochemical methods, including linear sweep voltammetry, cyclic voltammetry, and capacitance spectroscopy. Finally, the constructed immunosensor was used for analyzing TSH levels spiked in the artificial serum samples.