BODIPY based fluorescent turn-on sensor for highly selective detection of HNO and the application in living cells

On the basis of BODIPY platform, a terpyridyl-substituent BODIPY-Copper complex (Cu(II)-BTPY) was rationally designed and synthesized as a redox reaction fluorescent sensor for detecting HNO over reactive oxygen species (ROS) and reactive nitrogen species (RNS) with impressive selectivity in living cells under mild and neutral conditions. The BTPY exhibits relatively high fluorescence quantum efficiency as much as 34.8% and presents large stokes shift, about 62 nm. When a series of transition metal ions were exploited to investigate the fluorescence quench towards BTPY, copper ion (Cu²⁺) gave the optimal result. After the fluorescence of the probe being effectively quenched in the presence of Cu²⁺, it can be in turn recovered through the reduction of Cu²⁺ into Cu⁺ by HNO accompanying with a visually observable fluorescence response. Still, the sensing mechanism was evidently confirmed by EPR and ESI-MS measurement. In addition, the employment of BTPY for imaging dyes was also presented in vivo.     

基于离子液体-石墨烯-二茂铁的高灵敏电化学免疫传感器检测CEA

研究了在PBS缓冲介质中,一种检测癌胚抗原的新型免标记免疫电化学传感器的制备,将石墨烯、二茂铁的高效催化及壳聚糖的优良生物相容性和成膜性、离子液体的导电性等优势充分结合构建了电化学免疫传感器。通过循环伏安法及交流阻抗对修饰的电极进行表征,在最优条件下,癌胚抗原的质量浓度在0.2～50.0 ng/mL的范围内与差分脉冲伏安法峰电流呈良好的线性关系,回归方程为Δi=0.38-1.31ρ,相关系数分别为0.9967,检测限为0.06 ng/mL,该传感器可用于人血清样品的测定。     

Highly sensitive electrochemical impedance spectroscopic detection of DNA hybridization based on Aunano-CNT/PANnano films

A polyaniline nanofibers (PAN_nano)/carbon paste electrode (CPE) was prepared via dopping PAN_nano in the carbon paste. The nanogold (Au_nano) and carbon nanotubes (CNT) composite nanoparticles were bound on the surface of the PAN_nano/CPE. The immobilization and hybridization of the DNA probe on the Au_nano-CNT/PAN_nano films were investigated with differential pulse voltammetry (DPV) and cyclic voltammetry (CV) using methylene blue (MB) as indicator, and electrochemical impedance spectroscopy (EIS) using [Fe(CN)₆]^3−/4− as redox probe. The voltammetric peak currents of MB increased dramatically owing to the immobilization of the probe DNA on the Au_nano-CNT/PAN_nano films, and then decreased obviously owing to the hybridization of the DNA probe with the complementary single-stranded DNA (cDNA). The electron transfer resistance (R_et) of the electrode surface increased after the immobilization of the probe DNA on the Au_nano-CNT/PAN_nano films and rose further after the hybridization of the probe DNA. The remarkable difference between the R_et value at the DNA-immobilized electrode and that at the hybridized electrode could be used for the label-free EIS detection of the target DNA. The loading of the DNA probe on Au_nano-CNT/PAN_nano films was greatly enhanced and the sensitivity for the target DNA detection was markedly improved. The sequence-specific DNA of phosphinothricin acetyltransferase (PAT) gene and the polymerase chain reaction (PCR) amplification of nopaline synthase (NOS) gene from transgenically modified beans were determined with this label-free EIS DNA detection method. The dynamic range for detecting the PAT gene sequence was from 1.0 × 10⁻¹² mol/L to 1.0 × 10⁻⁶ mol/L with a detection limit of 5.6 × 10⁻¹³ mol/L.     

Highly sensitive electrochemical stripping detection of hepatitis B surface antigen based on copper-enhanced gold nanoparticle tags and magnetic nanoparticles

On the basis of copper-enhanced gold nanoparticle tags as an amplification approach, we introduced, in this paper, magnetic nanoparticles for further improving performance of electrochemical immunoassay by anodic stripping voltammetry (ASV) at a glassy-carbon electrode. Due to the use of antibody-immobilized magnetic nanoparticles, the immunoreaction between antibody and antigen takes place in a homogeneous bulk solution phase. Compared with traditional solid interface reaction, the proposed strategy can provide some advantages such as easy of separation, shorter analytical time, wider linear range, and lower detection limit. It was also successfully applied to HBsAg determination in a linear range of 0.1-1500 ng mL⁻¹ with a detection limit of 87 pg mL⁻¹. The proposed analytical strategy holds good selectivity, sensitivity and repeatability and also great promise for the extended application in the fields of clinical diagnosis, bio-affinity assay and environmental monitoring.     

Highly sensitive detection of microRNA by chemiluminescence based on enzymatic polymerization

We have developed a new methodology for miRNA assay using chemiluminescence imaging by poly(U) polymerase catalyzed miRNA polymerization. This method is very sensitive with a 50 fM limit of detection, which is comparable to or better than current assay methods. Multiplex detection for miRNA can be easily realized by introducing different capture probes onto the biosensor array, which will make it highly versatile for various research purposes.     

Highly sensitive measurement of mepindolol in biological fluids using HPLC and electrochemical detection

Summary Mepindolol, a β-blocker agent, has an indolic structure which can undergo oxidation. A high-performance liquid chromatographic technique has been used to measure mepindolol in biological fluids using pindolol as the internal standard. The chromatography has been combined with electrochemical detection (coulometric detector). This method allows the determination of very low amounts of mepindolol with good precision and accuracy, the limit of quantification being 0.6 ng · ml⁻¹.     

Label-free electrochemical aptasensor constructed by layer-by-layer technology for sensitive and selective detection of cancer cells

Here, a cytosensor was constructed with ferrocene-appended poly(allylamine hydrochloride) (Fc-PAH) functionalized graphene (Fc-PAH-G), poly(sodium-p-styrenesulfonate) (PSS) and aptamer (AS1411) by layer-by-layer assembly technology. The hybrid nanocomposite Fc-PAH-G not only brings probes on the electrode and also promotes electron transfer between the probes and the substrate electrode. Meanwhile, LBL technology provides more effective probes to enhance amplified signal for improving the sensitivity of the detection. While AS1411 forming G-quardruplex structure and binding cancer cells, the current response of the sensing electrode decreased due to the insulating properties of cellular membrane. Differential pulse voltammetry (DPV) was performed to investigate the electrochemical detection of HeLa cells attributing to its sensitivity of the current signal change. The as-prepared aptasensor showed a high sensitivity and good stability, a widely detection range from 10 to 10⁶ cells/mL with a detection limit as low as 10 cells/mL for the detection of cancer cells.     

Highly sensitive detection of platelet-derived growth factor on a functionalized diamond surface using aptamer sandwich design

Aptamer-based fluorescence detection of platelet-derived growth factor (PDGF) on a functionalized diamond surface was demonstrated. In this work, a sandwich design based on the ability of PDGF to bind with aptamers at its two available binding sites was employed. It was found that this sandwich design approach significantly increases the fluorescence signal intensity, and thereby a very low detection limit of 4 pM was achieved. The effect of the ionic strength of MgCl(2) buffer solution was also investigated, and the most favourable binding for PDGF-BB occurred at a Mg(2+) concentration of 5.5 mM. Since the aptamers bind to the target PDGF with high affinity, fluorescence detection exhibited high selectivity towards different biomolecules. The high reproducibility of detection was confirmed by performing three cycles of measurements over a period of three days.     

Highly sensitive detection of cancer cells based on the DNA barcode assay and microcapillary electrophoretic analysis

Considering rarity of circulating tumor cells (CTCs) in human blood, the development of highly sensitive detection techniques for cancer cells is crucial for prediction, diagnosis, and prognosis of cancers. In this study, we propose an advanced cellular detection method by combining a biobarcode assay and microcapillary electrophoresis (μCE) technology. While the DNA biobarcode assay can provide ultrasensitive and multiplex detection platforms, the μCE chip can analyze barcode DNAs with high speed and accuracy according to the DNA size. We designed the barcode DNA size as 20 bp for indicating the expression of epithelial cell adhesion molecules (EpCAM) biomarkers and 30 bp for assigning CDX2 expression which is specific for colorectal cancer cells with addition to two bracket ladders (15 and 45 bp). Using MCF‐7 (breast cancer) and SW620 (colorectal cancer) as models, we conducted a biobarcode assay and analyzed the resultant biobarcode DNA on the μCE chip. We could detect the 20 bp CE peak in the electropherogram even with ten MCF‐7 and SW620 cells in a volume of 200 μL, thereby demonstrating the highly sensitive detection of cancer cells. We furthermore identified the type of colorectal cancer by observing two positive peaks (20 bp for EpCAM and 30 bp for CDX2) in the μCE analysis.     

Highly sensitive electrochemical determination of Sunset Yellow based on gold nanoparticles/graphene electrode

An electrochemical sensor was prepared using Au nanoparticles and reduced graphene successfully decorated on the glassy carbon electrode (Au/RGO/GCE) through an electrochemical method which was applied to detect Sunset Yellow (SY). The as-prepared electrode was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM) and electrochemical measurements. The results of cyclic voltammetry (CV) proved that Au/RGO/GCE had the highest catalytic activity for the oxidation of SY as compared with GCE, Au/GCE, and RGO/GCE. Differential pulse voltammetry (DPV) showed that the linear calibration curves for SY on Au/RGO/GCE in the range of 0.002 μM–109.14 μM, and the detection limit was estimated to be 2 nM (S/N = 3). These results suggested that the obtained Au/RGO/GCE was applied to detect SY with high sensitivity, low detection limit and good stability, which provided a promising future for the development of portable sensor in food additives.     

Highly sensitive electrochemical label-free aptasensor based on dual electrocatalytic amplification of Pt-AuNPs and HRP

In this work, a label-free electrochemical aptamer-based sensor (aptasensor) was constructed on account of the direct immobilization of redox probes on an electrode surface. For this proposed aptasensor, a gold nanoparticles (AuNPs)-coated electrode was firstly modified with redox probes-nickel hexacyanoferrates nanoparticles (NiHCFNPs) through chemisorption and electrostatic adsorption. Then, platinum-gold alloy nanoparticles (Pt-AuNPs) and horseradish peroxidase (HRP) were respectively assembled onto the modified electrode surface, which formed the multilayer films for amplifying the electrochemical signal of NiHCFNPs and immobilizing thiolated thrombin aptamers (TBAs). In the presence of target thrombin, the TBA on the multilayer could catch the thrombin onto the electrode surface, which resulted in a barrier for electro-transfer, leading to the decrease of the electrochemical signal of NiHCFNPs amplified by the Pt-AuNPs and HRP toward H(2)O(2). The proposed method avoided the redox probes labeling process, increased the amount of redox probes, and further amplified the electrochemical signal. Thus, the approach showed a high sensitivity and a wider linearity to thrombin in the range between 0.01 nM and 50 nM with a detection limit of 6.3 pM.     

基于多巴胺-量子点的比色免疫分析法对甲胎蛋白的高灵敏检测

基于抗原抗体识别特异性,以多巴胺-Mn/ZnS量子点(DA-QDs)为反应媒介,紫外可见分光光度计为检测手段,采用柠檬酸钠还原法制备金纳米颗粒(AuNPs),其表面用辣根过氧化物酶(HRP)和羊抗小鼠免疫球蛋白G (IgG)修饰获得功能化二抗(IgG-AuNPs-HRP)作为信号放大标签,构建一种简便高灵敏的检测方法....     

Highly sensitive colorimetric detection of NH3 based on Au@Ag@AgCl core-shell nanoparticles

As an important component of the atmosphere, ammonia (NH₃) plays a very important role in maintaining the balance of environment. However, it is also one of the most toxic gases that can cause damage to the human respiratory system and mucous membranes even at low concentrations. As such, development of highly sensitive and selective NH₃ sensors is of high significance for environmental monitoring and health maintenance. Herein, we have synthesized Au@Ag@AgCl core-shell nanoparticles (NPs) by oxidative etching and precipitating Au@Ag core-shell NPs using FeCl₃ and further used them as optical probes for the colorimetric detection of NH₃. The sensing mechanism is based on the fact that the etching of NH₃ on AgCl and Ag shell leads to the variations of ingredients and core-to-shell ratio of the Au@Ag@AgCl NPs, thereby inducing noticeable spectral and color changes. By replacing the outmost layer of Ag with AgCl, not only is the stability of the sensor against oxygen significantly enhanced, but also is the sensitivity of the method improved. The method exhibits good linear relationship for the detection of NH₃ from 0 to 5000 μmol/L with the limit of detection of 6.4 μmol/L. This method was successfully applied to the detection of simulated air polluted by NH₃, indicating its practical applicability for environmental monitoring. This method shows great potential for on-site NH₃ detection particularly in remote area, where a simple, fast, low-cost, and easy-to-handle method is highly desirable.     

Rapid and sensitive DNA target detection using enzyme amplified electrochemical detection based on microchip

A rapid and sensitive DNA targets detection using enzyme amplified electrochemical detection (ED) based on microchip was described. We employed a biotin‐modified DNA, which reacted with avidin‐conjugated horseradish peroxidase (avidin–HRP) to obtain the HRP‐labeled DNA probe and hybridized with its complementary target. After hybridization, the mixture containing dsDNA‐HRP, excess ssDNA‐HRP, and remaining avidin–HRP was separated by MCE. The separations were performed at a separation voltage of +1.6 kV and were completed in less than 100 s. The HRP was used as catalytic labels to catalyze H₂O₂/o‐aminophenol reaction. Target DNA could be detected by the HRP‐catalyzed reduction with ED. With this protocol, the limits of quantification for the hybridization assay of 21‐ and 39‐mer DNA fragments were of 8×10^?12 M and 1.2×10^?11 M, respectively. The proposed method has been applied satisfactorily in the analysis of Escherichia coli genomic DNA. We selected the detection of PCR amplifications from the gene of E. coli to test the real applicability of our method. By using an asymmetric PCR protocol, we obtained ssDNA targets of 148 bp that could be directly hybridized by the single‐stranded probe and detected with ED.     

Highly sensitive electrochemical sensor for estradiol based on the signal amplification strategy of Cu-BDC frameworks

Using 1,4-Benzenedicarboxylic acid (H₂BDC) as the ligand, a kind of copper-based metal-organic frameworks (MOFs) were prepared and characterized using transmission electron microscopy, scanning electron microscopy, infrared spectroscopy, and X-ray diffraction. After that, the prepared Cu-BDC frameworks were used to modify the carbon paste electrode, constructing a novel electrochemical sensor for estradiol (E2). The prepared Cu-BDC frameworks are much more active for the oxidation of E2, and greatly increase the oxidation signals of E2. The results from chronocoulometry indicate that the Cu-BDC frameworks modified electrode exhibit much higher accumulation efficiency toward E2. Based on the signal amplification strategy of Cu-BDC frameworks, a sensitive and rapid electrochemical method was developed for the determination of E2. The linear range was from 5.00 to 650.0 nM, and the detection limit was as low as 3.80 nM. It was used in different water samples, and the values of recovery were over the range from 96.5 to 101%. The practical applications reveal that this new sensing system is accurate and convenient, and has great potential applications in the environmental monitoring.     

Highly sensitive detection of protein and small molecules based on aptamer-modified electrochemiluminescence nanoprobe

Amplified optical detection of biomolecules using nanoparticle as the carrier has attracted considerable interest in the scientific community. In this study, a promising aptasensor was developed for highly sensitive detection of protein and small molecules based on the construction of aptamer-modified electrochemiluminescence (ECL) nanoprobe. Specifically, thrombin and ATP serve as the examples for detection. By taking advantage of sandwich binding of two affinity aptamers for high specificity, tris-(2,2'-bipyridyl)ruthenium (TBR)-cysteamine loaded in gold nanoparticle (GNP) as barcodes for signal amplification, and micromagnetic particles (MMPs) based ECL technology for rapid detection, a novel assay for biomolecules quantification was developed. The sandwich complex containing targets could be selectively captured by MMPs and then quantified by ECL intensity. We have demonstrated that the detection limits of human thrombin and ATP are 1 pM and 10 pM, respectively, with high specificity. The proposed technology is expected to become a powerful tool for biomolecule analysis.     

Green electrochemical sensors based on boron-doped diamond and silver amalgam for sensitive voltammetric determination of herbicide metamitron

Monatshefte für Chemie - Chemical Monthly - Novel sensitive voltammetric methods for determination of herbicide metamitron were developed using polished and mercury meniscus-modified silver...     

DNA-directed immobilization of horseradish peroxidase-DNA conjugates on microelectrode arrays: towards electrochemical screening of enzyme libraries

This work is aimed towards the generation of enzyme arrays on electrochemically active surfaces by taking advantage of the DNA-directed immobilization (DDI) technique. To this end, two different types of horseradish peroxidase (HRP)-DNA conjugates were prepared, either by covalent coupling with a bifunctional cross-linker or by the reconstitution of apo-HRP, that is, HRP lacking its prosthetic heme (protoporphyrin IX) group, with a covalently DNA-modified heme cofactor. Both conjugates were characterized in bulk and also subsequent to their immobilization on gold electrodes through specific DNA hybridization. Electrochemical measurements by using the phenolic mediator ortho-phenylendiamine indicated that, due to the high degree of conformational orientation, the apparent Michaelis-Menten constants of the reconstituted HRP conjugate were lower than those of the covalent conjugate. Due to the reversible nature of DDI, both conjugates could be readily removed from the electrode surface by simple washing and, subsequently, the electrodes could be reloaded with fresh enzymes, thereby restoring the initial amperometric-response activity. Moreover, the specific DNA hybridization allowed us to direct the two conjugates to distinct sites on a microelectrode array. Therefore, the self-assembly and regeneration capabilities of this approach should open the door to the generation of arrays of redox-enzyme devices for the screening of enzymes and their effectors.     

Highly sensitive flow detection of uric acid based on an intermediate regeneration of uricase

The principle of the signal amplification of a uric acid sensor based on dithiothreitol (DTT)-mediated intermediate regeneration of uricase was applied to a flow-injection system with an immobilized uricase reactor and a DTT-containing carrier. Highly sensitive detection for nM to microM order of uric acid was achieved when 10 mM TRIS-HCl buffer (pH 10.0) containing 20 mM DTT was used as a carrier at 0.6 ml min-1 and 37 degrees C. The sensitivity of the uric acid was much improved over a batch method using a uricase membrane-coupling electrode, and the detection limit (ca. peak current 8 nA) of uric acid was found to be down to 3 x 10(-10) M (amplification factor; more than 10,000). This chemically amplified flow-system is very useful for the direct assay of uric acid in highly diluted biological fluids (urine and serum) without complicated pretreatment of the samples, because this sensor has the potential to detect trace amounts (nM to microM) of uric acid in highly diluted body fluids in which the concentration of interfering constituents was decreased to negligible levels. Good correlation was observed between this system and conventional spectrophotometry. The immobilized uricase reactor could be re-used for at least 4 months of repeated analysis without loss of activity and was stable if stored at 4 degrees C in 10 mM TRIS-HCl buffer, pH 9.0.