免标记比色核酸适配体传感器同步快速检测孔雀石绿和无色孔雀石绿

研究以双特异性核酸适配体A3作为传感探针、纳米金（AuNPs）为指示剂、NaCl溶液为聚集诱导剂,构建了一种新型的免标记AuNPs比色生物传感器,可实现水产品中孔雀石绿（MG）和无色孔雀石绿（LMG）的同步、快速、可视化检测。该方法的检测原理是核酸适配体A3对MG和LMG有双特异性识别能力,可作为MG和LMG理想的识别受体。它可通过静电作用吸附到AuNPs表面,保护AuNPs并抑制高盐溶液诱导的聚集,AuNPs溶液颜色不变,即为红色;当加入靶标MG或LMG后,该核酸适配体能够与靶标特异性结合,并从AuNPs表面上解离,AuNPs失去保护作用而在高盐溶液诱导下发生聚集,溶液颜色由红变蓝。根据颜色变化,可通过肉眼定性或通过光谱仪定量分析MG和LMG的残留量。该方法首先将50 μL的核酸适配体A3（终浓度150 nmol/L）与150 μL的AuNPs（终浓度1.25 nmol/L）混合,室温孵育6 min。随后加入50 μL待测液,室温孵育30 min。最后加入50 μL NaCl（终浓度150 mmol/L）,4 min后观察溶液颜色变化,并分别测定MG和LMG在520 nm和650 nm下的吸光度值。结果表明,在最佳反应条件下,该方法能够特异性检测MG和LMG,而对磺胺嘧啶（SDZ）和硝基呋喃妥因（NFT）无交叉反应;当MG、LMG的浓度为0~17.5 μmol/L时,吸光度比值与靶标浓度呈现良好的线性关系,相关系数（R ² ）分别为0.9938和0.9715。MG和LMG的检出限分别为6.93 nmol/L和6.38 nmol/L,加标回收率分别为88.60%~93.30%和101.80%~107.00%,相对标准偏差（RSD）分别为2.27%~3.55%和2.62%~3.75%。该方法操作简单,快速和灵敏,可为水产品中MG和LMG的同步快速检测提供一种新方法。     

Selection of a DNA aptamer for the development of fluorescent aptasensor for carbaryl detection

An improved ssDNA library immobilized systematic evolution of ligands by enrichment(SELEX) was applied to select aptamers against carbaryl.After nine selection rounds,a highly enriched ssDNA pool was obtained.The Apta3 was demonstrated as the optimal aptame r.In order to facilitate the modification of aptamer,the Apta3 was further truncated with the dissociation constant(K_d) of 0.3 64 ± 0.055 μmol/L and a fluorescent aptasensor was developed.The linear range for carbaryl was from 100 nmol/L to1500 nmol/L,with the limit of detection was as low as 15.23 nmol/L.Besides,the biosensor was validated for the carbaryl spiked real samples,and the recoveries were between 97.7% and 107.3%.     

A novel colorimetric aptasensor for detection of chloramphenicol based on lanthanum ion–assisted gold nanoparticle aggregation and smartphone imaging

A label-free, rapid response colorimetric aptasensor for sensitive detection of chloramphenicol (CAP) was proposed, which was based on the strategy of ssDNA-modified gold nanoparticle (AuNP) aggregation assisted by lanthanum (La³⁺) ions. The AuNPs generated a color change that could be monitored in the red, green, and blue and analyzed by the smartphone imaging app. La³⁺, as a trigger agent, strongly combined with the phosphate groups of the surface of ssDNA-AuNPs probe, which helps create AuNP aggregation and the color change of AuNPs from red to blue. On the contrary, when mixing with CAP, the aptamer (Apt) bound to CAP to form a rigid structure of the Apt-CAP complex, and La³⁺ attached to the phosphate groups of the complex, which prevented the aptamer from binding to the surface of the AuNPs. As a result, the color of the AuNPs changed to violet-red. Finally, UV-vis absorption spectroscopy and the smartphone imaging app were employed to determine CAP with a lower detection limit of 7.65 nM and 5.88 nM, respectively. The proposed strategy featuring high selectivity and strong anti-interference ability for detection of CAP in practical samples was achieved. It is worth mentioning that the simple and portable colorimetric aptasensor will be used for facilitating on-site detection of food samples.

     

Electrochemical impedance spectroscopy detection of lysozyme based on electrodeposited gold nanoparticles

A simple, highly sensitive, and label-free electrochemical impedance spectroscopy (EIS) aptasensor based on an anti-lysozyme-aptamer as a molecular recognition element, was developed for the detection of lysozyme. Improvement in sensitivity was achieved by utilizing gold nanoparticles (AuNPs), which were electrodeposited onto the surface of a gold electrode, as a platform for immobilization of the aptamer. To quantify the amount of lysozyme, changes in the interfacial electron transfer resistance (R_et) of the aptasensor were monitored using the redox couple of an [Fe(CN)₆]^3−/4− probe. The R_et increased with lysozyme concentration. The plot of R_et against the logarithm of lysozyme concentration is linear over the range from 0.1 pM to 500 pM with a detection limit of 0.01 pM. The aptasensor also showed good selectivity for lysozyme without being affected by the presence of other proteins.     

Au/In2O3 Nanocubes Based Label-free Aptasensor for Ultrasensitive and Rapid Recognition of Cardiac Troponin I

Cardiac Troponin I (cTnI) is a preferred biomarker to diagnose acute myocardial infarction which is one of the leading risks to health globally due to its short term. However, clinical analyzers are difficult to achieve its on-site quantitative detection. A novel label-free aptasensor was constructed to realize ultrasensitive and rapid recognition of cTnI. A nanocubic AuNPs/In₂O₃ composite was designed to provide synergistic effects of abundant active sites and signal magnification for aptamers grafting. Relying on a conductance-dependence strategy, this aptasensor can achieve the quantitative detection within 10 min, which is much faster than state-of-the-art analyzers, as well as exhibiting an ultrawide linear range of 0.1–1000 ng/mL and a low detection limit of 0.06 ng/mL with an excellent selectivity in the analysis of human serum.     

A Sandwich‐type Electrochemiluminescence Aptasensor for Thrombin Based on Functional Co‐polymer Electrode Using Ru(bpy)32+ Doped Nanocomposites as Signal‐amplifying Tags

Herein, a signal‐on sandwich‐type electrochemiluminescence (ECL) aptasensor for the detection of thrombin (TB) was proposed. The graphene (GR) doped thionine (TH) was electropolymerized synchronously on the bare glassy carbon electrode (GCE) to form co‐polymer (PTG) electrode. The gold nanoparticles (AuNPs) were decorated on the surface of the PTG by in‐situ electrodeposition, and the functional co‐polymer (PTG‐AuNPs) electrode was utilized as sensing interface. Then, TB binding aptamer I (TBA I) as capture probes were modified on the PTG‐AuNPs electrode to capture TB, and Ru(bpy)₃²⁺/silver nanoparticles doped silica core‐shell nanocomposites‐labeled TB binding aptamer II (RuAg/SiO₂NPs@TBA II) were used as signal probes to further bind TB, resulting in a sandwich structure. With the assistant of silica shell and AgNPs, the enrichment and luminous efficiency of Ru(bpy)₃²⁺ were significantly improved. Under the synergy of PTG‐AuNPs and RuAg/SiO₂NPs, the ECL signal was dramatically increased. The proposed ECL aptasensor displayed a wide linear range from 2 fM to 2 pM with the detection limit of 1 fM, which is comparable or better than that in reported ECL aptasensors for TB using Ru(bpy)₃²⁺ and its derivatives as the luminescent substance. The excellent sensitivity makes the proposed aptasensor a promising potential in pharmaceutical and clinical analysis.     

A novel electrochemiluminescence aptasensor for protein based on a sensitive N-(aminobutyl)-N-ethylisoluminol-functionalized gold nanoprobe

A novel electrochemiluminescence (ECL) aptasensor for platelet-derived growth factor B chain (PDGF-BB) assay was developed by assembling N-(aminobutyl)-N-ethylisoluminol functionalized gold nanoparticles (ABEI-AuNPs) with aptamers as nanoprobes. In the protocol, the biotinylated aptamer capture probes were first immobilized on a streptavidin coated gold nanoparticle (AuNPs) modified electrode, afterwards, the target PDGF-BB and the ABEI-AuNPs tagged aptamer signal probe were successively attached to the modified electrode by virtue of the dimer structure of PDGF-BB to fabricate a "sandwich" conjugate modified electrode, i.e. an aptasensor. ECL measurement was carried out with a double-step potential in carbonate buffer solution containing H(2)O(2). The aptasensor showed high sensitivity and selectivity toward PDGF-BB and specificity toward PDGF-BB aptamer. The detection limit was as low as 2.7 × 10(-14) M. In this work, the ABEI-AuNPs synthesized by a simple seed growth method have been successfully used as aptamer labels, which greatly amplified the ECL signal by binding numbers of ABEI molecules on the surface of AuNPs. The ABEI-AuNPs signal amplification is superior to other reported signal amplification strategies based on aptamer-related polymerase chain reaction or functionalized nanoparticles in simplicity, stability, labeling property and practical applicability. And the ABEI-AuNPs based nanoprobe is more sensitive than the luminol functionalized AuNPs based nanoprobe. Moreover, such an ultra-sensitive and low-cost assay can be accomplished with a simple and fast procedure by using a simple ECL instrumentation. The aptasensor was also applied for the detection of PDGF-BB in human serum samples, showing great application potential. Given these advantages, the ECL aptasensor is well suited for the direct, sensitive and rapid detection of protein in complex clinical samples.     

基于巯基嘌呤/多肽修饰的金纳米粒子的比色方法检测溶液中的镉离子

研究了一种基于双配体（巯基嘌呤（MP）和多肽CALNN）修饰金纳米粒子（AuNPs）的比色方法,用于快速、选择性地检测水溶液中的Cd2+。 其中,MP作为功能配体通过N原子与Cd2+发生配合作用,从而引起AuNPs聚集;CALNN配体有助于提高体系的稳定性和选择性。 当体系中无Cd2+时,溶液呈红色,随着Cd2+浓度的增加,溶液颜色逐渐由红色变为蓝紫色,这种颜色变化可以通过光谱测定还可以用肉眼直接观察。 该方法操作简便,具有较好的选择性和较快的响应速度（＜5 min）,其检测限达到350 nmol/L。     

Au Nanoparticle-DNAzyme Dual Catalyst System for Sensitively Colorimetric Detection of Thrombin

A novel colorimetric aptasensor was developed for thrombin detection with high sensitivity and specificity. The assay takes the advantage of Au nanoparticles-DNAzyme as a dual catalytic system for signal amplification. Au nanparticles were modified with peroxidase mimicking DNAzyme sequence as well as thrombin binding aptamer. And then the thrombin binding aptamer hybridized with its complementary sequence which was immobilized on the surface of the magnetic nanoparticles to construct the colorimetric aptasensor. In the presence of thrombin, the target-induced displacement takes place, resulting in the dissociation of the aptasensor. The DNAzyme functioned Au NPs are released due to the combine ability of thrombin binding aptamer with thrombin. The released Au NPs are capable of catalyzing the colorless 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid(ABTS) conversion into a blue-green product by H₂O₂-mediated oxidation, thus can amplify the colorimetric readout signals of thrombin detection. Such a device can serve as a novel selectivity sensor for thrombin with a detection limit of 0.6 nmol/L.     

Electrochemical aptasensor for detection of copper based on a reagentless signal-on architecture and amplification by gold nanoparticles

A highly sensitive and specific electrochemical aptasensor for Cu(2+) detection based on gold nanoparticles (AuNPs) is presented. In this work, AuNPs offered a big surface area to immobilize a large number of aptamers and excellent electrochemical signal transduction. Its high sensitivity, low detection limit, and wide detection range are the main advantages over our former copper aptasensor. The peak current increased proportionally to the Cu(2+) concentration over the range from 0.1 nM to 10 μM with a detection limit of 0.1 pM. The presence of other divalent metal ions did not affect the detection of Cu(2+), which indicates a high specificity of Cu(2+) detection could be detected. Rapidity, simplicity, and excellent selectivity make it suitable for practical use in determination of Cu(2+) from lake samples.     

Label‐free Electrochemical Aptasensor for Carcino‐embryonic Antigen Based on Ternary Nanocomposite of Gold Nanoparticles,Hemin and Graphene

In this report, a label‐free electrochemical aptasensor for carcino‐embryonic antigen (CEA) was successfully developed based on a ternary nanocomposite of gold nanoparticles, hemin and graphene nanosheets (AuNPs‐HGNs). This nanocomposite was prepared by decorating gold nanoparticles on the surface of hemin functionalized graphene nanosheets via a simple wet‐chemical strategy. The aptamer can be assembled on the surface of AuNPs‐HGNs/GCE (glassy carbon electrode) through Au‐S covalent bond to form the sensing interface. Hemin absorbed on the graphene nanosheets not only acts as a protective agent of graphene sheets, but also as an in situ probe base on its excellent redox properties. Gold nanoparticles provide with both numerous binding sites for loading CEA binding aptamer (CBA) and good conductivity to promote the electron transfer. The current changes, which are caused by CEA specifically binding on the modified electrode, are exploited for the label‐free detection of CEA in a very rapid and convenient protocol. Therefore, the method has advantages of high sensitivity, wide linear range (0.0001–10 ng mL^?1), low detection limit (40 fg mL^?1) and attractive specificity. The results illustrate that the proposed label‐free electrochemical aptasensor has a potential application in the biological or clinical target analysis for its simple operation and low cost.     

An intriguing electrochemical impedance aptasensor based on a porous organic framework supported silver nanoparticles for ultrasensitively detecting theophylline

Porous organic frameworks (POFs) are excellently stable porous materials, which can be employed as host platforms to support metal nanoparticles as functional composites for various applications. Herein, a novel POF is successfully prepared via Friedel-Crafts reaction. Silver nanoparticles (Ag NPs) are embedded in the prepared POF to generate an Ag@POF composite, which not only possesses high surface area, outstanding physicochemical stability and outstretched π-conjugation skeleton, but also exhibits preferable electrochemical stability and conductivity. This composite is able to immobilize a mass of aptamer strands to fabricate an intriguing electrochemical aptasensor. Electrochemical impedance spectroscopy (EIS) is a commonly used technology to analyze the electrochemical signal variation. The Ag@POF-based biosensor shows the excellent electrochemical detection behavior through analyzing EIS. For instance theophylline as a research mode, the Ag@POF based electrochemical aptasensor reveals ultra-sensitiveness, high selectivity, remarkable stability, good repeatability and simple operability even in various real samples. Notably, this aptasensor has the sensitive detection performance with the limit of detection of 0.191 pg/mL (1.06 pmol/L) in a wide concentration range of 5.0 × 10^-4 – 5.0 ng/mL (2.78 × 10^-3 – 27.8 nmol/L).     

Colorimetric recognition of melamine in milk using novel pincer zinc complex stabilized gold nanoparticles

A convenient colorimetric approach for visual detection of melamine in raw milk was realized by using gold nanoparticles (AuNPs) stabilized by an unsymmetrical terpyridyl zinc complex with a thymine fragment at one terminal and a quaternary ammonium salt at the other. Even without pre-addition of melamine or relative additives, obvious color change from red to blue was observed by naked eye in the presence of trace amount of melamine, which was attributed to the alternation of aggregation state of AuNPs caused by the selective binding between the thymine fragment and melamine via triple hydrogen-bonding interactions. Remarkably, the detection limit for melamine was as low as 2.4 ppb, providing a highly sensitive and efficient approach for the visual detection of melamine.     

A highly selective and sensitive cocaine aptasensor based on covalent attachment of the aptamer-functionalized AuNPs onto nanocomposite as the support platform

Based on the conformational changes of the aptamer-functionalized gold nanoparticles (AuNPs) onto MWCNTs/IL/Chit nanocomposite as the support platform, we have developed a sensitive and selective electrochemical aptasensor for the detection of cocaine. The 5′-amine-3′-AuNP terminated aptamer is covalently attached to a MWCNTs/IL/Chit nanocomposite. The interaction of cocaine with the aptamer functionalized AuNP caused the aptamer to be folded and the AuNPs with negative charge at the end of the aptamer came to the near of electrode surface therefore, the electron transfer between ferricyanide (K₃Fe(CN)₆) as redox probe and electrode surface was inhibited. A decreased current of (K₃Fe(CN)₆) was monitored by differential pulse voltammetry technique. In an optimized condition the calibration curve for cocaine concentration was linear up to 11 μM with detection limit (signal-to-noise ratio of 3) of 100 pM. To test the selectivity of the prepared aptasensor sensing platform applicability, some analgesic drugs as the interferes were examined. The potential of the aptasensor was successfully applied for measuring cocaine concentration in human blood serum. Based on our experiments it can be said that the present method is absolutely beneficial in developing other electrochemical aptasensor.     

DNA Electrochemical Aptasensor for Detecting Fumonisins B1 Based on Graphene and Thionine Nanocomposite

In this paper, a novel aptasensor was designed by with the dual amplification of Au nanoparticles (AuNPs) and graphene/thionine nanocomposites (GS‐TH) for sensitive determination of fumonisins B₁ (FB₁). AuNPs is modified at the electrode surface to increase the electrical conductivity and fabricate specific recognition interface for FB₁ through the hybridization of capture DNA and its aptamer. Large number of TH molecules were loaded at the surface of graphene sheet to served as electrochemical probe and increase its electrochemical signal due to the excellent conductivity and large surface area of graphene sheet. This type of nanocomposites is then assembled to the single strand section of FB₁ aptamer at electrode surface by π–π stacking interactions between them, leading to an enhanced electrochemical signal. After the specific combination between FB₁ aptamer and its target (FB₁) in solution, GS–TH was released from electrode surface, resulting in a decreased electrochemical signal. The result demonstrated that the decreased currents were proportional to the FB₁ concentration in the range of 1–10⁶ pg/mL with a detection limit of 1 pg/mL. Besides, the developed aptasensor was also applied successfully for the determination of FB₁ in feed samples. The result shows this aptasensor has a higher sensitivity and selectivity.     

Colorimetric detection of potassium ions using aptamer-functionalized gold nanoparticles

Herein, a simple and novel colorimetric method for detection of potassium ions (K⁺) was developed. The colorimetric experiments revealed that upon the addition of K⁺, the conformation of anti-K⁺ aptamer in solution changed from random coil structure to compact rigid G-quadruplex one. This compact rigid G-quadruplex structure could not protect AuNPs against K⁺-induced aggregation, and thus the visible color change from wine-red to blue-purple could be observed by the naked eye. The linear range of the colorimetric aptasensor covered a large variation of K⁺ concentration from 5 nM to 1 μM and the detection limit of 5 nM was obtained. Moreover, this assay was able to detect K⁺ with high selectivity and had great potential applications.     

Colorimetric determination of ofloxacin using unmodified aptamers and the aggregation of gold nanoparticles

A colorimetric method is presented for the determination of the antibiotic ofloxacin (OFL) in aqueous solution. It is based on the use of an aptamer and gold nanoparticles (AuNPs). In the absence of OFL, the AuNPs are wrapped by the aptamer and maintain dispersed even at the high NaCl concentrations. The solution with colloidally dispersed AuNPs remains red and has an absorption peak at 520 nm. In the presence of OFL, it will bind to the aptamer which is then released from the AuNPs. Hence, AuNPs will aggregate in the salt solution, and color gradually turns to blue, with a new absorption peak at 650 nm. This convenient and specific colorimetric assay for OFL has a linear response in the 20 to 400 nM OFL concentration range and a 3.4 nM detection limit. The method has a large application potential for OFL detection in environmental and biological samples.

Open image in new window

Graphical abstract Schematic of a sensitive and simple colorimetric aptasensor for ofloxacin (OFL) detection in tap water and synthesic urine. The assay is based on the salt-induced aggregation of gold nanoparticles which results in a color change from red to purple.

     

Amplified impedimetric aptasensor based on gold nanoparticles covalently bound graphene sheet for the picomolar detection of ochratoxin A

An amplified electrochemical impedimetric aptasensor for ochratoxin A (OTA) was developed with picomolar sensitivity. A facile route to fabricate gold nanoparticles covalently bound reduced graphene oxide (AuNPs–rGO) resulted in a large number of well-dispersed AuNPs on graphene sheets with tremendous binding sites for DNA, since the single rGO sheet and each AuNP can be loaded with hundreds of DNA strands. An aptasensor with sandwich model was fabricated which involved thiolated capture DNA immobilized on a gold electrode to capture the aptamer, then the sensing interface was incubated with OTA at a desired concentration, followed by AuNPs–rGO functionalized reporter DNA hybridized with the residual aptamers. By exploiting the AuNPs–rGO as an excellent signal amplified platform, a single hybridization event between aptamer and reporter DNA was translated into more than 10⁷ redox events, leading to a substantial increase in charge-transfer resistance (R_ct) by 7∼ orders of magnitude compared with that of the free aptamer modified electrode. Such designed aptasensor showed a decreased response of R_ct to the increase of OTA concentrations over a wide range of 1 pg mL⁻¹–50 ng mL⁻¹ and could detect extremely low OTA concentration, namely, 0.3 pg mL⁻¹ or 0.74 pM, which was much lower than that of most other existed impedimetric aptasensors. The signal amplification platform presented here would provide a promising model for the aptamer-based detection with a direct impedimetric method.     

Simple and sensitive colorimetric detection of cysteine based on ssDNA-stabilized gold nanoparticles

In this paper, we demonstrate a simple and sensitive colorimetric detection of cysteine based on the cysteine-mediated color change of ssDNA-stabilized gold nanoparticles (AuNPs). Cysteine is capable of absorbing onto AuNPs surfaces via the strong interaction between its thiol group and gold. ssDNA molecules which stabilize AuNPs against salt-induced aggregation are removed away by cysteine encapsulation on the AuNPs surfaces, resulting in a characteristic color change of AuNPs from red to blue as soon as salt is added. The ratio of absorptions at 640 to 525 nm (A ₆₄₀/A ₅₂₅) is linear dependent on the cysteine concentration in the range from 0.1 to 5 μM. Furthermore, amino acids other than cysteine cannot mediate the color change under the identical conditions due to the absence of thiol groups, suggesting the selectivity of the proposed method toward cysteine. The employment of complicated protocols and sophisticated processes such as the preparation of modified AuNPs are successfully avoided in design to realize the simple and low-cost cysteine detection; and the high sensitivity and low cost of the method is favorable for practical applications. Figure In the presence of cysteine, cysteine binds to the AuNPs surface via Au-S bond, spontaneously driving ssDNA molecules away from the nanoparticles, which leads to the AuNPs aggregation under the condition of NaCl introduction, and the corresponding color change from red to blue. However, the presence of other amino acids results in no color change due to the absence of thiol groups. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Aptamer-wrapped gold nanoparticles for the colorimetric detection of omethoate

Organophosphorous pesticide(OP) contamination has serious adverse effects on human health and the environment. Due to the toxicity of OPs and the threat presented by their accidental or intentional release in populated areas, the determination and monitoring of these OPs in food products and environment is of great importance. OPs are present in very small quantities and therefore, methods for their detection need to be highly sensitive and selective. Here, we aimed to develop a simple and selective aptamer-based colorimetric assay for the detection of omethoate, which is one of the commonly used OPs. The principle of the assay is that single-stranded DNA(ss DNA)-wrapped gold nanoparticles(Au NPs) are resistant to salt-induced aggregation. By employing an "artificial antibody" organophosphorous pesticide-binding aptamer(OBA) as the recognition element, aptamer-wrapped Au NPs(Au-apta) show high selectivity towards omethoate, resulting in the disconnection of aptamers from Au NPs and the aggregation of Au NPs. As there is a significant color change from the interparticle plasmon coupling during the aggregation of Au NPs, the established assay showed good linearity between 0.1 and 10 μmol/L, with a low detection limit of 0.1 μmol/L. Other OPs such as profenofos, phorate, and isocarbophos would not interfere with the detection of omethoate despite having similar structures. Thus, the colorimetric method shows potential for use in the detection of omethoate in real soil samples.