A graphene-based real-time fluorescent assay of deoxyribonuclease I activity and inhibition

Using the remarkable difference in the affinity of graphene oxide (GO) with double strand DNA (dsDNA) and short DNA fragments, we report for the first time a GO-based nonrestriction nuclease responsive system. Our system was composed of GO and a fluorescent dye fluorescein amidite (FAM)-labeled dsDNA substrate (F-dsDNA). At first, the fluorescence of this F-dsDNA substrate was quenched upon addition of GO. When nuclease was added to the mixture of dsDNA and GO, hydrolysis of dsDNA was initiated and small DNA fragments were produced. As a result, the short FAM-linked DNA fragments were released from GO due to the weak affinity of GO with short DNA fragments, and the fluorescence got a restoration. At present, many sensing systems are based on the fact that GO prefers to bind long single strand DNA (ssDNA) over dsDNA or short ssDNA. As for our system, GO has a prior binding with dsDNA over short DNA fragments. Compared with previous methods, this assay platform has some advantages. First, since GO can be prepared in large quantities from graphite available at very low cost, this method shows advantages of simplicity and cost efficiency. Besides, the proposed GO-based nuclease assay provides high sensitivity due to the super quenching capacity of GO. Using deoxyribonuclease I (DNase I) as a model system, DNase I activity can be quantitatively analyzed by the velocity of the enzymatic reaction, and 1.75 U mL⁻¹ DNase I can be significantly detected. Moreover, the fluorescent intensity with various concentrations of nuclease becomes highly discriminating after 3–8 min. Thus, it is possible to detect nuclease activity within 3–8 min, which demonstrates another advantage of quick response of the present system. Finally, use of dsDNA as substrate, our method can achieve real-time nuclease activity/inhibition assay, which is time-saving and effortless.     

Importance of membrane selection in the development of immunochromatographic assays for low-molecular weight compounds

This study was performed to demonstrate the importance of selecting an appropriate membrane when developing immunochromatographic assays (ICAs) for the sensitive detection of low-molecular weight compounds. Based on our findings, we propose a theoretical basis for selecting such a membrane. When eluting the sample solution for the competitive ICA using colloidal gold label for low-molecular analytes, the degree of binding inhibition is proportional to the collision frequency between the antibody-colloidal gold (Ab–CG) and analyte before Ab–CG binding to the capture antigen and a higher concentration of pesticides around the Ab–CG leads to a greater degree of inhibition. Therefore, we propose that the relative migration speed of the analyte and Ab–CG on the test strip is critically important for selecting a membrane in the development of sensitive competitive ICAs. We developed a novel method to estimate such a relative migration speed. We demonstrated the applicability of this proposal by using it to select an appropriate membrane for the development of an ICA of the pesticide diazinon.     

Biotin-Streptavidin-enhanced Enzyme-Linked Immunosorbent Assay for the Determination of Parathion-Methyl in Vegetables

The development of rapid, simple, and sensitive analytical methods for food contaminants is a topic of considerable interest. Polyclonal antibody against parathion-methyl (PM) was raised and used to develop a biotin-streptavidin indirect competitive enzyme-linked immunosorbent assay (BS-icELISA) with improved sensitivity for the determination of PM in vegetable. At the optimum conditions, the IC₅₀ and limit of detection of BS-icELISA for PM were found to be 1.2 µg/L and 0.2 µg/L, respectively, which is 6-fold more sensitive than the traditional icELISA. This method was applied to determine PM residue in three vegetable samples with a simple and effective extraction procedure, good recoveries (in the range of 88.2–108.7% and with coefficients of variation below 15%) and accuracy (correlation coefficient of 0.9576 with GC-MS) were obtained. Our results indicated that the biotin-streptavidin system is useful in improving the sensitivity of ELISA and could be used for routine monitoring of pesticide at trace level.     

A multiplex single nucleotide polymorphism genotyping method using ligase‐based mismatch discrimination and CE‐SSCP

Accuracy, simplicity, and cost‐effectiveness are the most important criteria for a genotyping method for SNPs compatible with clinical use. One method developed for SNP genotyping, ligase‐based discrimination, is considered the simplest for clinical diagnosis. However, multiplex assays using this method are limited by the detection method. Although CE has been introduced as an alternative to error prone microarray‐based detection, the design process and multiplex assay procedure are complicated because of the DNA size‐dependent separation principle. In this study, we developed a simple and accurate multiplex genotyping method using reaction condition‐optimized ligation and high‐resolution CE‐based SSCP. With this high‐resolution CE‐SSCP system, we are able to use similar‐sized probes, thereby eliminating the complex probe design step and simplifying the optimization process. We found that this method could accurately discriminate single‐base mismatches in SNPs of the tp53 gene, used as targets for multiplex detection.     

Portable paper‐based device for quantitative colorimetric assays relying on light reflectance principle

This paper presents a novel paper‐based analytical device based on the colorimetric paper assays through its light reflectance. The device is portable, low cost (<20 dollars), and lightweight (only 176 g) that is available to assess the cost‐effectiveness and appropriateness of the original health care or on‐site detection information. Based on the light reflectance principle, the signal can be obtained directly, stably and user‐friendly in our device. We demonstrated the utility and broad applicability of this technique with measurements of different biological and pollution target samples (BSA, glucose, Fe, and nitrite). Moreover, the real samples of Fe (II) and nitrite in the local tap water were successfully analyzed, and compared with the standard UV absorption method, the quantitative results showed good performance, reproducibility, and reliability. This device could provide quantitative information very conveniently and show great potential to broad fields of resource‐limited analysis, medical diagnostics, and on‐site environmental detection.     

中药致肾毒性成分马兜铃酸A单抗制备及酶联免疫分析方法的建立

采用活化酯法,将马兜铃酸A分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联,得到免疫抗原马兜铃酸A-BSA和包被抗原马兜铃酸A-OVA.利用马兜铃酸A-BSA免疫Bal b/c小鼠,制得鼠单克隆抗体1A11,单抗效价为2×104;单抗为IgG1类,轻链为κ型;与其结构类似物马兜铃酸B、C和D的交叉反应率分别为2.8%,3.5%和31.2%.基于抗马兜铃酸A单克隆抗体的间接竞争酶联免疫分析方法(icELISA)的IC50为1.9 μg/L,检测范围为0.5～7.5 μg/L.icELISA添加回收率为86%～97%,相对标准偏差在5.2%～11.1%之间.利用所建立的icELISA测定了6个中药材和5个中成药中马兜铃酸A的含量,并用高效液相色谱法(HPLC)进行了验证,其中关木通、广防己、天仙藤、马兜铃和青木香中均检测出马兜铃酸A,而川木通和5个中成药中未检测到马兜铃酸A.结果表明: 本方法可用于中药中马兜铃酸A的快速检测.     

MOLECULAR ENGINEERING STUDIES ON NONTHROMBOGENIC BIOMATERIALS--A NOVEL CLASS OF NONTHROMBOGENIC BIOMATERIALS WITH ZWITTERIONIC STRUCTURE OF CARBOXYBETAINES

N,N-dimethyl-N-methacryloyloxyethyl-N-carboxyethyl ammonium (DMMCA) was graft-copolymerized onto the surface of segmented poly(ether urethane) (SPEU) and PE film. The carboxybetaine structure on SPEU and PE film surfaces was confirmed by ATR-FTIR, XPS and water contact angle measurements. Through the experiments with platelet adhesion and protein adhesion assay in vitro, the two materials studied, including poly-DMMCA gel, all show excellent nonthrombogenicity. This confirms once again that the zwitterionic molecular structure on the surfaces of materials is essential for improving their nonthrombogenicity and biocompatibility.     

Beyond labels: A review of the application of quantum dots as integrated components of assays, bioprobes, and biosensors utilizing optical transduction

A comprehensive review of the development of assays, bioprobes, and biosensors using quantum dots (QDs) as integrated components is presented. In contrast to a QD that is selectively introduced as a label, an integrated QD is one that is present in a system throughout a bioanalysis, and simultaneously has a role in transduction and as a scaffold for biorecognition. Through a diverse array of coatings and bioconjugation strategies, it is possible to use QDs as a scaffold for biorecognition events. The modulation of QD luminescence provides the opportunity for the transduction of these events via fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), charge transfer quenching, and electrochemiluminescence (ECL). An overview of the basic concepts and principles underlying the use of QDs with each of these transduction methods is provided, along with many examples of their application in biological sensing. The latter include: the detection of small molecules using enzyme-linked methods, or using aptamers as affinity probes; the detection of proteins via immunoassays or aptamers; nucleic acid hybridization assays; and assays for protease or nuclease activity. Strategies for multiplexed detection are highlighted among these examples. Although the majority of developments to date have been in vitro, QD-based methods for ex vivo biological sensing are emerging. Some special attention is given to the development of solid-phase assays, which offer certain advantages over their solution-phase counterparts.     

Kinetics of comet formation in single‐cell gel electrophoresis: Loops and fragments

We investigated the mechanisms of DNA exit during single‐cell gel electrophoresis (the comet assay) by measuring the kinetics of the comet tail formation. In the neutral comet assay, the rate of DNA exit was found to be dependent on the topological state of DNA, which was influenced by either ethidium bromide or a low radiation dose. The results clearly show that the comet tail is formed by extended DNA loops: the loop extension, being reversible when the DNA torsional constraint remains in the loops, is favored when the constraint is relaxed. The kinetics of the comet formation in the case of a high radiation dose points out that accumulation of the single‐strand breaks causes DNA fragmentation. In contrast to the neutral comet assay, the alkaline comet assay is not related to the chromatin loops. Our results imply that the alkaline treatment induces detachment of the loops from the nuclear matrix, and the comet tail is formed by ssDNA fragments, the ends of which are pulled out from the comet head by electric force. We suggest that the kinetic approach can be considered as an important improvement of the comet assay.     

Development of liposomal immunosensor for the measurement of insulin with femtomole detection

The monitoring of insulin is of great relevance for the management of diabetes, the detection of pancreatic islet-cell malfunction, the definition of hypoglycemia, and the diagnosis of insulinoma. A liposomal immunosensing system for the determination of insulin was developed in this study. The insulin sensor was constructed by the immobilization of anti-insulin antibodies on the inner wall of the microcapillary immunoseparator. Liposomes tagged with anti-insulin and encapsulating a fluorescent dye were used as the detectable label. In the presence of insulin, sandwich immunocomplexes were formed between the immobilized antibodies in the column, the sample of insulin, and the antibody-tagged sulforhodamine B-dye-loaded liposomes. Signals generated by lysing the bound liposomes with 30 mM n-octyl-β-d-glucopyranoside were measured by a fluorescence detector. The detected signal was directly proportional to the amount of insulin in the test sample. The liposomal immunosensing system successfully detected as low as 136 attomole. MeOH (30%) was used for the regeneration of antibody-binding sites in the microcapillary after each measurement, which allowed the immunoseparator to be used for at least 70 repeated assays. The antibody activity in this proposed microcapillary immunoseparator could be well maintained for at least 1 week. The calibration curve for insulin in Tris-buffered saline had a linear dynamic range of 10 pM-10 nM, and the total assay time was less than 30 min. The coefficient of variation for triplicate measurements was <5.00%, which indicated that well-reproducible results can be obtained by this newly developed method.