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Aptamer-functionalized Nanoparticles for the Detection of Platelet-derived Growth Factor and Immunoglobulin G
No full text蛋白質檢測極為重要，並可應用於臨床診斷、癌症、病原體感染和遺傳性疾病的研究。我們利用比色法開發出兩種具有高選擇性和高靈敏度的蛋白質生物感測器，並使用磁性奈米粒子與兩種金奈米粒子—核酸適體修飾的13奈米金粒子(Apt–Au NPs)，和纖維蛋白原吸附的金奈米粒子(Fib–Au NPs, 56 nm)等來檢測血小板生長因子(platelet-derived growth factors, PDGF)以及人類免疫球蛋白G (hIgG)。在檢測PDGF的系統中，Apt–Au NPs有辨識目標分子的功能，而Fib–Au NPs則是扮演reporting units。在此系統中，功能性金奈米粒子(AptPDGF/Aptthr29–Au NPs)上面有與血小板生長因子結合的核酸適體(PDGF-binding-aptamer, AptPDGF)，以及與凝血酶結合的核酸適體(thrombin-binding-aptamer, Aptthr29)。凝血酶會催化修飾在金奈米上的血纖維蛋白原，形成不溶的纖維素，因此造成了金奈米粒子的聚集，若凝血酶的活性被AptPDGF/Aptthr29–Au NPs抑制，則不會造成聚集。如果溶液中有PDGF先與AptPDGF/Aptthr29–Au NPs結合，則凝血酶就因立體障礙而無法接至AptPDGF/Aptthr29–Au NPs表面。此系統偵測PDGF的線性範圍是0.5–20 nM (R2 = 0.96)，在100 μM的牛血清蛋白存在下，偵測極限是0.3 nM。若把 AptPDGF/Aptthr29–Au NPs用來濃縮樣品中的PDGF，那麼偵測極限可降至35 pM。最後我們測定乳癌細胞培養基中的PDGF濃度為230 (±20) pM，證明此方法是一個簡單，具有高專一性和靈敏度的方法。第二個主題是將類似的概念應用於hIgG的檢測。第一步先將hIgG與修飾protein G (PG)的磁性奈米粒子(PG–MNPs)還有PG修飾的Apt–Au NPs (PGApt–Au NPs)結合，並用磁鐵分離它們的複合體(PG–MNPs…hIgG…PGApt–Au NPs)。第二步是利用上清液中沒有被磁鐵分離的PGApt–Au NPs來控制凝血酶的活性，因此越多hIgG，會形成越多的PG–MNPs…hIgG…PGApt–Au NPs複合體，上清液中和凝血酶結合的PGApt–Au NPs也就越少，使fibrin–Au NPs的聚集越嚴重。在最佳化的條件並且含有100 μM的牛血清蛋白之下，PG–MNPs/PGApt–Au NPs/Fib–Au NPs probe可將偵測極限降至5 nM 。
我們最後取得三個正常人的血液樣本與兩個類風濕關節炎病人的血液樣本，利用標準曲線測得其hIgG濃度，並和市售的儀器做比較(enzyme-linked immunosorbent assay)，發現兩者的數值呈現良好的線性(R2 = 0.98)，證明了此法的實用性與準確性。Protein detection is of great importance in basic research and clinical diagnosis of genetic disorders and its associated diseases, cancers, and pathogen infections. We have developed two colorimetric protein sensors using aptamer modified 13-nm gold nanoparticles (Apt–Au NPs), thrombin, and fibrinogen adsorbed Au NPs (Fib–Au NPs; 56 nm). These could be used for the highly selective and sensitive detection of platelet-derived growth factors (PDGFs) and human immunoglobulin G (hIgG). In the PDGF system, Apt–Au NPs and Fib–Au NPs were the recognition and reporting units, respectively. PDGF-binding-aptamer (AptPDGF) and 29-base-long thrombin-binding-aptamer (Aptthr29) were conjugated with Au NPs to prepare functional Apt–Au NPs (AptPDGF/Aptthr29–Au NPs) for specific interaction with PDGF and thrombin, respectively. Thrombin interacted with Fib–Au NPs in solution to catalyze the formation of insoluble fibrillar fibrin–Au NPs agglutinates through the polymerization of unconjugated and conjugated fibrinogen. Thrombin activity was suppressed when it interacted with AptPDGF/Aptthr29–Au NPs due to steric effects through the specific interaction of PDGF with AptPDGF on the surfaces of AptPDGF/Aptthr29–Au NPs. Under optimal conditions with AptPDGF/Aptthr29–Au NPs at 25 pM, thrombin at 400 pM, and Fib–Au NPs at 30 pM, AptPDGF/Aptthr29–Au NPs/Fib–Au NPs probe responded linearly to PDGF over a concentration range of 0.5–20 nM with a correlation coefficient of 0.96. The limit of detection (LOD, signal-to-noise ratio = 3) for each of the three PDGF isoforms was 0.3 nM in the presence of bovine serum albumin at 100 μM. When using AptPDGF/Aptthr29–Au NPs to selectively enrich PDGF and remove interfering substances from cell media, LOD of this probe for PDGF was 35 pM. This probe revealed that the concentration of PDGF in the three cell media is 230 (±20) pM, showing its advantages in terms of simplicity, sensitivity, and specificity. We also developed a method for the selective
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and sensitive detection of human immunoglobulin G (hIgG). The first step involves the specific interactions of hIgG with protein G (PG)-functional Fe2O3 magnetic NPs (PG–MNPs) and with PG– and aptamer (Apt)– modified gold NPs (PGApt–Au NPs) and the subsequent magnetic separation of their complexes (PG–MNPs…hIgG…PGApt–Au NPs). In the second step, the concentration of free PGApt–Au NPs was determined by taking advantage of their control of thrombin activity toward fibrinogen-modified Au NPs (Fib–Au NPs). The activity of thrombin toward Fib–Au NPs to form fibrin–Au NP aggregates was inhibited by PGApt–Au NPs through the specific interaction of thrombin with the Apt. The greater the amount of hIgG in a sample, the less free PGApt–Au NPs remained in the supernatant. Consequently, greater amounts of free thrombin remained, which led to the formation of greater amounts of fibrin–Au NP aggregates. Under optimal conditions (8 μg/mL PG–MNPs, 1.0 nM PGApt–Au NPs, 400 pM thrombin, 30 pM Fib–Au NPs), PG–MNPs/PGApt–Au NPs/Fib–Au NPs probe allows the selective detection of hIgG down to 5 nM in the presence of 100 μM of BSA. The practicality of this approach was validated by determining the concentrations of hIgG in spiked plasma samples that were in good agreement with determinations made by enzyme-linked immunosorbent assays (R2 = 0.98). These results demonstrate that this assay has great potential for diagnosing diseases associated with changes in hIgG levels
Mapping the antioxidant activity of apple peels with soft probe scanning electrochemical microscopy
Full text linkWe present a non-invasive electrochemical strategy for mapping the antioxidant (AO) activity of apple peels, which counterbalances oxidative stress caused by various external effectors. Soft carbon microelectrodes were used for soft probe scanning electrochemical microscopy (SECM) enabling the gentle and scratch-free in contact mode scanning of the rough and delicate apple peels in an electrolyte solution. The SECM feedback mode was applied using ferrocene methanol (FcMeOH) as redox mediator that gets electrochemically oxidized at the soft probe and diffuses towards the apple peel where it gets regenerated by certain AOs leading to a redox mediator recycling and increased current signal. The global AO activity in the apple peel including lenticels and regions with artificially degraded AOs were mapped using the soft microelectrodes. Finally, in an apple cross-section the higher and homogeneous AO concentration in the peel with a heterogeneously decaying AO gradient towards the apple inward was visualized, demonstrating the adequate micrometer resolution of the SECM probe and the possibility to get information of the interior AO activity of the apple.LEP
Colorimetric detection of platelet-derived growth factors through competitive interactions between proteins and functional gold nanoparticles
No full textElectrochemical Imaging of Cells and Tissues
No full textThe technological and experimental progress in electrochemical imaging of biological specimens is discussed with a view on potential applications for skin cancer diagnostics, reproductive medicine and microbial testing. The electrochemical analysis of single cell activity inside cell cultures, 3D cellular aggregates and microtissues is based on the selective detection of electroactive species involved in biological functions. Electrochemical imaging strategies, based on nano/micrometric probes scanning over the sample and sensor array chips, respectively, can be made sensitive and selective without being affected by optical interferences as many other microscopy techniques. The recent developments in microfabrication, electronics and cell culturing/tissue engineering have evolved in affordable and fast-sampling electrochemical imaging platforms. We believe that the topics discussed herein demonstrate the applicability of electrochemical imaging devices in many areas related to cellular functions.LEP
Multiple scanning electrochemical microscopy mapping of tyrosinase in micro-contact printed fruit samples on polyvinylidene fluoride membrane
Full text linkHerein, we introduce three orthogonal and compatible methods for detecting tyrosinase, a key factor in fruit browning and skin disorders, with high spatial resolution by means of scanning electrochemical microscopy (SECM). All methods are performed subsequently on the same substrate area providing a wide range of relevant information. The first SECM approach that relies on the mapping of a differential pore oxygen concentration induced by the local hydrophobic changes that the adsorption of tyrosinase generates on a porous polyvinylidene fluoride (PVDF) membrane. The second approach is based on the direct monitoring of the enzymatic activity of tyrosinase by detecting amperometrically the reaction products from the enzymatic conversion of L-3,4-dihydroxyphenylalanine (L-DOPA). Finally, tyrosinase was visualized implementing a tyrosinase sandwich immunoassay readout by SECM. The multiple SECM detection strategies were successfully applied to map unequivocally the tyrosinase enzymatic activity of a micro-contact printed banana sample. Furthermore, differential pulse voltammetry and mass spectrometry analyses were employed to elucidate the nature of the electrochemical response obtained during the tyrosinase enzymatic activity experiments.LEP
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Microfluidic Probes for Scanning Electrochemical Microscopy
No full textThis chapter discusses the soft microfluidic scanning electrochemical microscopy (SECM) probes represent a versatile tool for supporting various investigations ranging from the electrochemical characterization of different surfaces to the local perturbation of the microenvironment of adherently grown live cells. Microfluidic SECM probes were electrochemically characterized by cyclic voltammetry (CV) in a redox mediator solution delivered through the microfluidic channel. SECM in the feedback mode has been employed for the imaging of human fingerprints based on the detection of silver nanoparticles or benzoquinone‐tagged proteins. The application of the microfluidic SECM probes as a tool for spatiotemporal live cell perturbations has a significant potential toward biological applications, for monitoring in vitro hypoxic tumor models toward the discovery of new therapeutic compounds and the understanding of effects induced by localized pH changes within malignant cells. The microfluidic SECM probes can be easily multiplexed in order to investigate large surfaces or increase the capacity toward drug screening
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