影响双(2-丁氧羰基-3,4,6-三氯苯基)草酸酯化学发光体系的因素

过氧草酸酯类化学发光是一类以多氯代水杨酸酯与草酰氯反应生成的草酸酯、过氧化氢和荧光剂为主要组成的化学发光体［１，２］．它除了具有发光效率高，强度大，寿命长的特点外，还可以通过选择荧光剂调节发光的颜色．采用这类发光体系可制成多种化学光源．在草酸酯和荧光...     

链酚胺型配体锌(Ⅱ)配合物模拟碳酸酐酶研究

合成了酚胺型链状配体,N,N'-二(2-羟基苄基)丙二胺(H~2L)及其Zn(Ⅱ)配合物(ZnL),通过元素分析、IR和^1HNRM等手段进行了表征。采用pH电位滴定法,在25℃±0.1℃,I=0.1mol·dm^-^3(KNO~3)条件下,测定了配体的质子化常数以及配体与金属离子Zn(Ⅱ)配位反应平衡常数。讨论了配体与金属离子Zn(Ⅱ)的配位情况,得到了配位酚羟基的解离常数。运用分光光度法,在25℃±0.1℃,I=0.1mol·dm^-^3(KNO~3)条件下,在pH=5.5～9.0(50mmol·dm^-^3缓冲溶液)范围内,研究了配合物作为碳酸酐酶模拟物催化对-硝基苯酚乙酸酯(NA)水解动力学,得到了NA酯水解的配合物催化速率常数k~N~P。实验结果表明,ZnHL^+的配位酚羟基的解离常数pK~a为6.83;催化速率常数k~N~P与pH之间不存在Sigmoidal型曲线关系,而是在pH值中性附近有最大值,ZnHL^+对NA酯水解有很好的催化效果,并且采取双重催化机理,是碳酸酐酶很好的模拟物。     

测定核黄素的化学发光新方法

研究了核黄素的化学发光性质，建立了一种测定核黄素的化学发光新方法，并应用于实际样品中核黄素的测定，与文献相比，本方法无需避光，而且分析体系和样品处理简单；干扰小，牛血清白蛋白、抗坏血酸、天冬氨酸等８种生物有机物质和 Ｃｕ（Ⅱ）、Ｚｎ（Ⅱ）、Ｆｅ（Ⅲ）等１５种常见金属离子的允许量较大；核黄素溶液浓度在０． １０～５０ ｍｇ／Ｌ范围内，与化学发光强度成正比，方法线性范围宽；测定１．０ｍｇ／Ｌ核黄素溶液１２次，求得相对标准偏差为２．９％，方法精密度高；方法的检测限为０．０７５ ｍｇ／Ｌ，灵敏度高。     

Battery-triggered ultrasensitive electrochemiluminescence detection on microfluidic paper-based immunodevice based on dual-signal amplification strategy

Dual-signal amplification strategy for ultrasensitive electrochemiluminescence (ECL) multiplexed immunoassay on microfluidic paper-based analytical devices (μ-PADs) was demonstrated. This dual-signal amplification technique was achieved by employing graphene oxide-chitosan/gold nanoparticles (GCA) immunosensing platform and [4,4′-(2,5-dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl) dibenzoic acid] (P-acid) functionalized nanoporous silver (P-acid/NPS) signal amplification label. For further low-cost and disposable applications, battery-triggered constant-potential ECL (+1.0 V for P-acid label (vs. Ag/AgCl auxiliary electrode)) was applied on this paper-based immunodevice with the aid of a home-made voltage-tunable power device, allowing the traditional electrochemical workstation to be abandoned. We found that two tumor markers could be sequentially detected in the linear ranges of 0.003–20 and 0.001–10 ng mL⁻¹ with the detection limits down to 1.0 and 0.8 pg mL⁻¹, respectively, by simply reversing the connection mode on two working electrodes. The results exhibited excellent precision and high sensitivity of such immunoassay, and it also demonstrated that this battery-triggered ECL paper-based immunodevice could provide a rapid, simple and simultaneous multiplex immunoassay with high throughput, low-cost and low detection limits for point-of-care testing.     

Open-sandwich immunoassay for sensitive and broad-range detection of a shellfish toxin gonyautoxin

At present, the analytical method for paralytic shellfish poisoning (PSP) toxins in shellfish is the mouse bioassay (MBA), which is an official method of the Association of Analytical Communities (AOAC [8]). However, the low sensitivity and concerns over the number of live animals required for testing have been cited as the major reason for seeking its replacement. In this report, we employed an open-sandwich immunoassay (OS-IA) to detect gonyautoxin (GTX2/3), a kind of PSP toxins. OS-IA, which utilizes the antigen-induced enhancement of antibody V_H/V_L interaction, can measure a small molecule antigen in a noncompetitive format. Hence it has a wider working range and shorter measurement time. We isolated anti-GTX2/3 antibody gene from a hybridoma GT-13A by screening a Fab-displaying phage library. Then the vectors for OS-IA were constructed, and examined for antigen concentration-dependency of the V_H/V_L interaction by OS-ELISA. As a result, in each case, signal intensity increases notably in a wide concentration range (0.1 to >1000 ng mL⁻¹) of free GTX2/3, which was enough to cover its regulation value (80 μg 100 g⁻¹) in many countries. So OS-IA will be widely applicable to detect PSP toxins in shellfish meats and in drinking water.     

Comparison of copper labeling followed by liquid chromatography-inductively coupled plasma mass spectrometry and immunochemical assays for serum hepcidin-25 determination

Hepcidin-25 has been defined as the key biomarker in iron metabolism. This peptide binds to the iron transporter ferroportin to cause its degradation. Therefore, the need for specific, accurate and precise methods for the quantification of hepcidin-25 in biological fluids is dramatically increasing. In this regard, the use of rapid immunochemical methods that provide low limit of quantification is desired for routine clinical use. However, such fast methodologies should be first analytically evaluated and compared with alternative strategies to check for their advantages and limitations. Here we compare the use of a commercial immunochemical assay for hepcidin determination with a novel analytical approach based on Cu-labeling of the peptide followed by Cu determination using liquid chromatography (HPLC) and plasma mass spectrometry (ICP-MS). The figures of merit of both systems reveal similar analytical characteristics and both seem to be adequate for the determination of the peptide at biologically relevant concentrations in human serum samples. The analysis of a larger number of samples (n = 50) by both techniques showed a good agreement in the concentrations found. Such finding permits to address the hepcidin recovery in the sample preparation procedure necessary for the HPLC-ICP-MS analysis in human serum that turn out to be 76–85%. Additionally, limitations due to cross-reactivity issues of the ELISA method could be addressed in some of the samples by using LC-ICP-MS and were confirmed by LC-Electrospray-MS.     

Ink‐Jet Microdispensing for the Formation of Gradients of Immobilised Enzyme Activity

Summary: The integration of gradients of enzyme activity in microstructured biosensor arrays enables intrinsic on‐line quality control of biosensor performance. Multiple sensor elements with different compositions and hence varying responses for the same analyte are evaluated as a basis for improving data reliability. The formation of glucose oxidase/polymer microstructures using a piezo microdispenser and their examination by scanning electrochemical microscopy (SECM) are used to demonstrate the feasibility of this approach.

Optical microscope image of grids obtained by dispensing of 1 mg/mL GOx and 2 mg/mL Vinnapas® mixture.     

Homocoenzyme B12 and Bishomocoenzyme B12: Covalent Structural Mimics for Homolyzed,Enzyme‐Bound Coenzyme B12

Efficient electrochemical syntheses of “homocoenzyme B₁₂” ( 2 , Co_β‐(5′‐deoxy‐5′‐adenosyl‐methyl)‐cob(III )alamin) and “bishomocoenzyme B₁₂” ( 3 , Co_β‐[2‐(5′‐deoxy‐5′‐adenosyl)‐ethyl]‐cob(III )alamin) are reported here. These syntheses have provided crystalline samples of 2 and 3 in 94 and 77 % yield, respectively. In addition, in‐depth investigations of the structures of 2 and 3 in solution were carried out and a high‐resolution crystal structure of 2 was obtained. The two homologues of coenzyme B₁₂ ( 2 and 3 ) are suggested to function as covalent structural mimics of the hypothetical enzyme‐bound “activated” (that is, “stretched” or even homolytically cleaved) states of the B₁₂ cofactor. From crude molecular models, the crucial distances from the corrin‐bound cobalt center to the C5′ atom of the (homo)adenosine moieties in 2 and 3 were estimated to be about 3.0 and 4.4 Å, respectively. These values are roughly the same as those found in the two “activated” forms of coenzyme B₁₂ in the crystal structure of glutamate mutase. Indeed, in the crystal structure of 2 , the cobalt center was observed to be at a distance of 2.99 Å from the C5′ atom of the homoadenosine moiety and the latter was found to be present in the unusual syn conformation. In solution, the organometallic moieties of 2 and 3 were shown to be rather flexible and to be considerably more dynamic than the equivalent group in coenzyme B₁₂. The homoadenosine moiety of 2 was indicated to occur in both the syn and the anti conformations.