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.     

Apoferritin protein nanoparticles dually labeled with aptamer and horseradish peroxidase as a sensing probe for thrombin detection

A novel and ultrasensitive sandwich-type electrochemical aptasensor has been developed for the detection of thrombin, based on dual signal-amplification using HRP and apoferritin. Core/shell Fe₃O₄/Au magnetic nanoparticles (AuMNPs) loading aptamer1 (Apt1) was used as recognition elements, and apoferritin dually labeled with Aptamer2 (Apt2) and HRP was used as a detection probe. Sandwich-type complex, Apt1/thrombin/Apt2–apoferritin NPs–HRP was formed by the affinity reactions between AuMNPs–Apt1, thrombin, and Apt2–apoferritin–HRP. The complex was anchored on a screen-printed carbon electrode (SPCE). Differential pulse voltammetry (DPV) was used to monitor the electrode response. The proposed aptasensor yielded a linear current response to thrombin concentrations over a broad range of 0.5–100 pM with a detection limit of 0.07 pM (S/N = 3). The detection signal was amplified by using apoferritin and HRP. This nanoparticle-based aptasensor offers a new method for rapid, sensitive, selective, and inexpensive quantification of thrombin, and offers a promising potential in protein detection and disease diagnosis.     

Sensitive Electrochemical Aptasensor for Thrombin Detection with Platinum Nanoparticles,Blocking Reagent‐Horseradish Peroxidase and Graphene Oxide as Enhancers

A highly sensitive thrombin electrochemical aptasensor with Pt nanoparticles, blocking reagent‐horseradish peroxidase (HRP) and inert graphene oxide (GO) as enhancers was successfully fabricated. Firstly, Pt nanoparticles with high surface to volume ratio could increase the amount of the immobilized redox probe hexacyanoferrate nanoparticles (NiHCFNPs) and effectively enhance the electron transfer. Secondly, HRP and Pt nanoparticles with high catalytic activity extremely amplify the electrochemical signal of NiHCFNPs toward H₂O₂. Lastly, inert graphene oxide (GO) labeled TBA could be used for enlarging the steric hindrance of thrombin. As a result, the aptasensor showed a high sensitivity with a detection limit of 500 fM.     

Amplified inhibition of the electrochemical signal of ferrocene by enzyme-functionalized graphene oxide nanoprobe for ultrasensitive immunoassay

A nanoprobe-induced signal inhibition mechanism was designed for ultrasensitive electrochemical immunoassay at a chitosan-ferrocene (CS-Fc) based immunosensor. The nanoprobe was prepared by covalently loading signal antibody and high-content horseradish peroxidase (HRP) on the graphene oxide (GO) nanocarrier. The immunosensor was prepared through the stepwise assembly of gold nanoparticles (Au NPs) and capture antibody at a CS-Fc modified electrode. After sandwich immunoreaction, the GO-HRP nanoprobes were quantitatively captured onto the immunosensor surface and thus induced the production of a layer of insoluble film through the enzymatically catalytic reaction of the HRP labels. Both the dielectric immunocomplex formed on the immunosensor surface and the enzymatic precipitate with low electroconductivity led to the electrochemical signal decease of the Fc indicator, which was greatly amplified by the multi-enzyme signal amplification of the nanoprobe. Based on this amplified signal inhibition mechanism, a new ultrasensitive electrochemical immunoassay method was developed. Using carcinoembryonic antigen as a model analyte, this method showed a wide linear range over 5 orders of magnitude with a detection limit down to 0.54 pg/mL. Besides, the immunosensor showed good specificity, acceptable reproducibility and stability as well as satisfactory reliability for the serum sample analysis.     

Platinum-gold alloy nanoparticles and horseradish peroxidase functionalized nanocomposite as a trace label for ultrasensitive electrochemical detection of thrombin

A novel tracer, platinum-gold alloy nanoparticles (Pt-AuNPs) and horseradish peroxidase (HRP) functionalized single-walled carbon nanotubes (SWCNTs) composite, is employed to label the secondary thrombin aptamer for constructing an ultrasensitive electrochemical aptasensor. Thionine, immobilized on functionalized SWCNTs, provides a pair of distinguished redox peak for electrochemical detection. Both the high-content Pt-AuNPs and HRP on SWCNTs amplify the electrochemical signal of thionine through electrocatalytic reduction of H(2)O(2). Differential pulse voltammetry (DPV) is employed to detect thrombin with different concentrations. The reduction peak current is logarithmically related to the concentration of thrombin in an extremely wide range from 10 fM to 5 nM with a detection limit of 3.6 fM. The dual signal amplification of Pt-AuNPs and HRP functionalized nanocomposite provides a promising way for ultrasensitive assay in electrochemical aptasensors.     

A microfluidic device based on a screen-printed carbon electrode with electrodeposited gold nanoparticles for the detection of IgG anti-Trypanosoma cruzi antibodies

In this article we report the development of an integrated microfluidic system coupled to a screen-printed carbon electrode (SPCE) applied to the quantitative determination of IgG specific antibodies present in serum samples of patients that suffer from Chagas disease. This relevant parasitic infection caused by the hemoflagellate protozoan Trypanosoma cruzi represents a major public health concern in Latin America. In order to perform the detection of mentioned antibodies, SPCE coupled to a microfluidic device was modified by electrodeposition of gold nanoparticles (AuNPs) and functionalized with Trypanosoma cruzi proteins from epimastigote membranes. The developed microfluidic immunosensor with immobilized T. cruzi proteins on the SPCE surface was successfully applied in the detection of specific IgG anti-T. cruzi antibodies, which were allowed to react immunologically with immobilized T. cruzi antigen. After that, labelled antibodies were quantified through the addition of horseradish peroxidase (HRP) enzyme-labeled secondary antibodies specific to human IgG, using 4-tert-butylcatechol (4-TBC) as enzymatic mediator. HRP in the presence of hydrogen peroxide (H(2)O(2)) catalyzes the oxidation of 4-TBC whose back electrochemical reduction was detected on a modified electrode at -100 mV. The calculated detection limit for electrochemical detection was 3.065 ng mL(-1) and the intra- and inter-assay coefficients of variation were below 6.95%.     

Rapid Legionella pneumophila determination based on a disposable core–shell Fe3O4@poly(dopamine) magnetic nanoparticles immunoplatform

A novel amperometric magnetoimmunoassay, based on the use of core–shell magnetic nanoparticles and screen-printed carbon electrodes, was developed for the selective determination of Legionella pneumophila SG1. A specific capture antibody (Ab) was linked to the poly(dopamine)–modified magnetic nanoparticles (MNPs@pDA-Ab) and incubated with bacteria. The captured bacteria were sandwiched using the antibody labeled with horseradish peroxidase (Ab-HRP), and the resulting MNPs@pDA-Ab-Legionella neumophila-Ab-HRP were captured by a magnetic field on the electrode surface. The amperometric response measured at −0.15 V vs. Ag pseudo-reference electrode of the SPCE after the addition of H₂O₂ in the presence of hydroquinone (HQ) was used as transduction signal. The achieved limit of detection, without pre-concentration or pre-enrichment steps, was 10⁴ Colony Forming Units (CFUs) mL⁻¹. The method showed a good selectivity and the MNPs@pDA-Ab exhibited a good stability during 30 days. The possibility of detecting L. pneumophila at 10 CFU mL⁻¹ level in less than 3 h, after performing a membrane-based preconcentration step, was also demonstrated.     

G‐Quadruplex‐Based DNAzymes Aptasensor for the Amplified Electrochemical Detection of Thrombin

A novel G‐quadruplex‐based DNAzymes aptasensor for the amplified electrochemical detection of thrombin has been described. The aptasensor utilized a combination of hemin and guanine‐rich thrombin‐binding aptamer (TBA) to form horseradish peroxidase (HRP)‐mimicking DNAzymes with peroxidase catalytic activity. In the presence of thrombin, the enzyme activity could be extensively promoted, thereby providing the amplified electrochemical readout signals for detecting thrombin. This aptasensor exhibited high sensitivity and selectivity for thrombin determination, which enabled the analysis of thrombin with a detection limit of 6×10^–11 M. On the basis of results, this method could have broad applications in the detection of proteins and other biomolecules.     

利用互补核酸杂交富集金胶实现信号扩增的电化学凝血酶蛋白生物传感器研究

介绍了一种利用互补核酸杂交富集金胶实现信号扩增的蛋白质生物传感器. 以凝血酶蛋白为研究对象, 利用凝血酶蛋白相对应的两段核酸适配体, 将适配体Ⅰ固定在磁性颗粒上, 用于特异性地捕获蛋白, 将适配体Ⅱ标记金胶作为检测信标. 由凝血酶蛋白和相对应的两段核酸适配体构建三明治结构的凝血酶蛋白生物传感器. 另外, 再通过信标金胶上过剩的核酸适配体链与另一段标记有金胶的互补核酸进一步杂交, 获得金胶的选择性聚集, 实现了信号扩增. 通过信号扩增, 使此传感器的灵敏度大大提高, 对凝血酶蛋白的检测下限可达到4.52×10-15 mol/L. 平行测定浓度为7.47×10-14 mol/L的凝血酶8次, 其RSD为3.0%. 该生物传感器对凝血酶蛋白有很好的特异性, 其它蛋白如溶菌酶和牛血清白蛋白的存在对于检测没有影响.     

Ultrasensitive electrochemical detection for thrombin using hybridization chain reaction with enzyme-amplification

In this work, a new electrochemical aptasensor using hybridization chain reaction (HCR) for signal amplification was developed for highly sensitive detection of thrombin. The sandwich system of aptamer/thrombin/aptamer–primer complex was fabricated as the sensing platform. As the initiator strands, aptamer–primer complex could propagate a chain reaction of hybridization events between the two hairpin probes, and whether long nicked DNA polymers could be formed on the modified electrode. Then the biotin-labeled dsDNA polymers could introduce numerous avidin-labeled horseradish peroxidase (HRP), resulting in significantly amplified electrochemical signal through the electrocatalysis of HRP. On the basis of the enzymatic oxidization of Fe²⁺ by H₂O₂ to yield Fe³⁺, the imaging of thrombin was detected by the reduction current of Fe³⁺ with the scanning electrochemical microscopic tip. The electrochemical signals had a good linear with logarithm of thrombin concentration in the range from 1.0 fM to 100 fM, reaching a detection limit of thrombin as low as 0.04 fM. In addition, the proposed strategy exhibited excellent specificity and was successfully applied in real sample assay which demonstrated the potential application in clinical diagnostics.     

Voltammetric determination of alkaline phosphatase and horseradish peroxidase activity using 3-indoxyl phosphate as substrate Application to enzyme immunoassay

The use of 3-indoxyl phosphate (3-IP) as an electrochemical substrate for ELISAs with voltammetric detection was investigated. Indirect measurements of alkaline phosphatase (AP) and horseradish peroxidase (HRP) activity in solution were carried out. Picomolar levels of both enzymes can be detected, which enables the design of electrochemical immunoassays using this substrate. The enzymatic turnover of the substrate gives indigo blue, insoluble in aqueous solutions. This product is easily converted into its soluble parent compound, indigo carmine (IC), by addition of fuming sulphuric acid to the reaction media. IC shows a reversible voltammetric peak at the formal potential of −0.15 V (versus Ag pseudo-reference electrode) when a screen-printed carbon electrode (SPCE) is used. The peak current of this process constitutes the analytical signal. Using this approach an ELISA assay to quantify pneumolysin (PLY, a toxin related to respiratory infections) was carried out using AP or HRP as enzymatic label. Calibration plots obtained are reported. 3-IP is demonstrated to be the first suitable substrate for the two most common enzyme labels used in immunoassays.     

A repeatable assembling and disassembling electrochemical aptamer cytosensor for ultrasensitive and highly selective detection of human liver cancer cells

In this work, a repeatable assembling and disassembling electrochemical aptamer cytosensor was proposed for the sensitive detection of human liver hepatocellular carcinoma cells (HepG2) based on a dual recognition and signal amplification strategy. A high-affinity thiolated TLS11a aptamer, covalently attached to a gold electrode through Au–thiol interactions, was adopted to recognize and capture the target HepG2 cells. Meanwhile, the G-quadruplex/hemin/aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (G-quadruplex/hemin/aptamer–AuNPs–HRP) nanoprobe was designed. It could be used for electrochemical cytosensing with specific recognition and enzymatic signal amplification of HRP and G-quadruplex/hemin HRP-mimicking DNAzyme. With the nanoprobes as recognizing probes, the HepG2 cancer cells were captured to fabricate an aptamer-cell-nanoprobes sandwich-like superstructure on a gold electrode surface. The proposed electrochemical cytosensor delivered a wide detection range from 1 × 10² to 1 × 10⁷ cells mL⁻¹ and high sensitivity with a low detection limit of 30 cells mL⁻¹. Furthermore, after the electrochemical detection, the activation potential of −0.9 to −1.7 V was performed to break Au–thiol bond and regenerate a bare gold electrode surface, while maintaining the good characteristic of being used repeatedly. The changes of gold electrode behavior after assembling and desorption processes were investigated by electrochemical impedance spectroscopy and cyclic voltammetry techniques. These results indicate that the cytosensor has great potential in disease diagnostic of cancers and opens new insight into the reusable gold electrode with repeatable assembling and disassembling in the electrochemical sensing.     

Electrochemical studies of a novel biosensor based on the CuO nanoparticles coated with horseradish peroxidase to determine the concentration of phenolic compounds

The present work is an attempt to design new electrochemical biosensor that easily determines the concentration of phenolic compounds in water and wastewaters. It is well-known that enzymes can add selectivity towards one or limited number of compounds. Thus, in the present study, horseradish peroxidase (HRP) was coated on CuO nanoparticles and immobilized on the electrode using a thin polymeric film. The electrochemical studies of modified electrode were carried out by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The voltammetric and EIS results confirm the possibility of HRP direct electron transfer at CuO nanoparticles. Our results revealed that the modified electrode can be used to determine the phenol concentration in the standard solutions and real samples. The present biosensor has good relative standard deviation (RSD% = 7%) and detection limit (DL of 0.1 μM), in addition to high stability.     

Fabrication of Bienzymatic Glucose Biosensor Based on Novel Gold Nanoparticles‐Bacteria Cellulose Nanofibers Nanocomposite

An exploration of gold nanoparticles–bacterial cellulose nanofibers (Au‐BC) nanocomposite as a platform for amperometric determination of glucose is presented. Two enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP) were immobilized in Au‐BC nanocomposite modified glassy carbon electrode at the same time. A sensitive and fast amperometric response to glucose was observed in the presence of electron mediator (HQ). Both of GOx and HRP kept their biocatalytic activities very well in Au‐BC nanocomposite. The detection limit for glucose in optimized conditions was as low as 2.3 µM with a linear range from 10 µM to 400 µM. The biosensor was successfully applied to the determination of glucose in human blood samples.     

Electrochemical Immunoassay for Cytomegalovirus Antigen Detection with Multiple Signal Amplification Using HRP and Pt‐Pd Nanoparticles Functionalized Single‐walled Carbon Nanohorns

Cytomegalovirus is typically associated with immunocompromised hosts, pregnant women and transplant patients, who require a timely diagnosis. In this work, a sensitive and highly specific electrochemical amplification immunosensor was established for detecting Cytomegalovirus pp65 antigen based on Pt‐PdNPs@SWCNHs with horseradish peroxidase (HRP) as a signal enhancer and thionine as a signal probe. First, Pt nanoparticle (PtNP) and Pd nanoparticle (PdNP) functionalized single‐walled carbon nanohorn (SWCNH) nanocomposites, i.e. Pt‐PdNPs@SWCNHs, was used as a carrier for immobilization of antibody through the Pt‐N bond and the Pd‐N bond. Next, HRP was used to block the rest of the binding‐sites. Signal amplification was obtained by the cooperative catalytic activities of Pt‐PdNPs and HRP to H₂O₂. SWCNHs loaded with a large amount of Pt‐PdNPs further amplified the signal due to the excellent surface area. The fabricated immunosensor was used to detect different concentrations of Cytomegalovirus pp65 antigen under optimized conditions. The tests showed a linear range from 0.1 to 80 ng mL^?1 with a low detection limit of 30 pg mL^?1, and exhibited excellent selectivity, stability and reproducibility. Therefore, this project presented a potential approach for the early diagnosis of Cytomegalovirus infection in clinical trials.     

基于DNA四面体纳米结构探针的电化学生物传感器检测DNA甲基化

该文以DNA四面体纳米结构探针(TSP)为捕获探针,将辣根过氧化物酶标记的IgG抗体结合在纳米金颗粒表面(AuNPs-IgG-HRP)作为信号分子,构建了一种新型DNA甲基化电化学传感器。利用一步热变性法组装成TSP后,通过Au—S键固定在修饰纳米金颗粒的金电极表面,经过靶标DNA杂交、5-甲基胞嘧啶(5-mc)抗体及AuNPs-IgG-HRP结合后,用差分脉冲伏安法(DPV)进行检测。采用循环伏安法(CV)和电化学阻抗谱(EIS)对修饰电极的构建过程进行电化学表征。探究了杂交时间、5-mc抗体浓度、IgG-HRP加入体积、氢醌(HQ)和过氧化氢(H₂O₂)浓度对传感器的影响。在最佳条件下,该传感器对甲基化DNA的线性响应范围为1.0×10^-15～1.0×10^-10 mol/L,检出限(S/N=3)为4.4×10^-16 mol/L。该传感器具有良好的选择性和稳定性,为DNA甲基化检测提供了新方法。     

Comparative Study of HRP,a Peroxidase‐Mimicking DNAzyme,and ALP as Enzyme Labels in Developing Electrochemical Genosensors for Pathogenic Bacteria

A comparative evaluation of an electrochemical sandwich genoassay for pathogenic bacteria based on immobilized hairpin DNA probes and three different enzyme labels (horseradish peroxidase, alkaline phosphatase and a biomimetic peroxidase‐like DNAzyme) is reported. The natural enzymes were used as streptavidin conjugates, coupled to the surface duplex by using a biotin‐labeled signaling probe, whereas the DNAzyme was directly incorporated to the sequence of the signaling probe. HRP provides enhanced sensitivity although the choice of a catalytic reporter DNA sequence could simplify the assay.     

An Electrochemical Aptasensor for Detection of Thrombin based on Target Protein‐induced Strand Displacement

A sensitive electrochemical aptasensor for detection of thrombin based on target protein‐induced strand displacement is presented. For this proposed aptasensor, dsDNA which was prepared by the hybridization reaction of the immobilized probe ssDNA (IP) containing thiol group and thrombin aptamer base sequence was initially immobilized on the Au electrode by self‐assembling via Au? S bind, and a single DNA labeled with CdS nanoparticles (DP‐CdS) was used as a detection probe. When the so prepared dsDNA modified Au electrode was immersed into a solution containing target protein and DP‐CdS, the aptamer in the dsDNA preferred to form G‐quarter structure with the present target protein resulting that the dsDNA sequence released one single strand and returned to IP strand which consequently hybridized with DP‐CdS. After dissolving the captured CdS particles from the electrode, a mercury‐film electrode was used for electrochemical detection of these Cd²⁺ ions which offered sensitive electrochemical signal transduction. The peak current of Cd²⁺ ions had a good linear relationship with the thrombin concentration in the range of 2.3×10^?9–2.3×10^?12 mol/L and the detection limit was 4.3×10^?13 mol/L of thrombin. The detection was also specific for thrombin without being affected by the coexistence of other proteins, such as BSA and lysozyme.     

An aptamer-based assay for thrombin via structure switch based on gold nanoparticles and magnetic nanoparticles

An aptamer-based assay for thrombin with high specificity and sensitivity was presented. In the protocol, the aptamer for thrombin was immobilized on magnetic nanoparticle, and its complementary oligonucleotide was labeled with gold nanoparticles, then the aptamer was hybridized with the complementary oligonucleotide to form the duplex structure as a probe, this probe could be used for the specific recognition for thrombin. In the presence of thrombin, the aptamer prefer to form the G-quarter structure with thrombin, resulting in the dissociation of the duplex of the probe and the release of the gold labeled oligonucleotide. Upon this, we were able to detect thrombin through the detection of the electrochemical signal of gold nanoparticles. The strategy combines with the high specificity of aptamer and the excellent characteristics of nanoparticles. This assay is simple, rapid, sensitive and highly specific, it does not require labeling of thrombin, and it could be applied to detect thrombin in complex real sample. The method shows great potential in other protein analysis and in disease diagnosis.     

A competitive immunochromatographic assay for testosterone based on electrochemical detection

An immunochromatographic assay using nitrocellulose membrane was combined with electrochemical detection using an electrode chip in order to quantitatively detect testosterone as a model analyte. The electrode chip consisted of a gold working electrode, a counter electrode and a pseudo-reference electrode, all fabricated on the bottom of a 3.2 mm × 3.2 mm well. Competitive immunoreactions on the membrane were initiated by flowing a solution containing testosterone and horseradish peroxidase (HRP)-labeled testosterone (a competitor) over the membrane. Prepared membrane was placed in a solution containing ferrocenemethanol (FcOH) and H₂O₂ in the well of the electrode chip, and the enzyme reaction was detected by amperometry. Labeled HRP captured on the membrane catalyzed the oxidation of FcOH to the oxidized form FcOH⁺, which was reduced electrochemically by the electrode chip. The electrochemical response of the reduction current decreased with increasing concentration of testosterone over the range 1–625 ng/ml.