Electro‐nano Diagnostic Platforms for Simultaneous Detection of Multiple Cancer Biomarkers

In this study, a bimetallic nanomaterial‐based electrochemical immunosensor was developed for the detection of carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF) cancer biomarkers at the same time. CEA and VEGF biomarkers are indicators for colon and breast cancers and stomach cancers, respectively. During the study, gold nanoparticle (AuNp), lead nanoparticle (PbNp), copper nanoparticle (CuNp) and magnetic gamma iron(III)oxide (γFe₂O₃ Np) were synthesized, characterized and used together for the first time in the structure of an electrochemical biosensor based on anti‐CEA and anti‐VEGF. For this purpose, Au SPE based sandwich immunosensor was fabricated by using labeled anti‐CEA (labeled with Pb⁺²) and labeled anti‐VEGF (labeled with Cu⁺²). As a result, CEA and VEGF biomarkers were detected following the oxidation peaks of label metals (Pb⁺² and Cu⁺²) by using differential pulse voltammetry. After the experimental parameters were optimized, the linear range was found in the concentration range between 25 ng/mL and 600 ng/mL with the relative standard deviation (RSD) value of (n=3 for 600 ng/mL) 3.33 % and limit of detection (LOD) value of 4.31 ng/mL for CEA biomarker. On the other hand, the linear range was found in the concentration range between 0.2 ng/mL and 12.5 ng/mL with the RSD value of (n=3 for 12.5 ng/mL) 5.31 % and LOD value of 0.014 ng/mL for VEGF biomarker. Lastly, sample application studies for synthetic plasma sample and interference studies with dopamine, ascorbic acid, BSA, cysteine and IgG were carried out.     

基于金纳米颗粒负载的金属有机骨架材料的癌胚抗原传感器的研制

以负载Au的金属有机骨架材料(AuNPs/Cu-TPA)标记CEA抗体(Ab2)为信号探针,通过电还原的方法将氧化石墨烯还原到电极上,研制了一种捕获CEA抗体(Ab1)的电化学免疫传感器,并将其应用于癌胚抗原(CEA)检测.所合成的MOFs材料中含有大量Cu2+,且电化学信号比较稳定,因此可以通过检测MOFs材料中Cu2+的信号实现对CEA的检测.此信号探针不需要预处理和酸处理,易负载贵金属从而固定抗体,大大简化了检测步骤并缩短了检测时间.此传感器对CEA的检测灵敏度好,操作简便.在最优实验条件下,此传感器的线性范围为0.1～ 80 ng/mL,检出限为0.03 ng/mL,线性相关系数为0.9887,可用于真实样品中CEA的测定.     

磁性纳米金共价固定癌胚抗原单克隆抗体的电流型免疫传感器

合成了Fe3O4/Au磁性复合纳米粒子, 在粒子表面通过自组装硫脲分子使表面氨基化, 再用戊二醛共价交联固定癌胚抗原抗体(anti-CEA). 在外加磁场的作用下, 将anti-CEA复合磁性粒子吸附在固体石蜡碳糊电极表面, 制成了新型电流型免疫传感器. 免疫电极在含有癌胚抗原CEA和辣根过氧化物酶标记的癌胚抗原(HRP-CEA)的混合溶液中温育, CEA和HRP-CEA与固定在电极表面的anti-CEA发生竞争反应, 导致HRP对H2O2的催化降解作用的改变, 从而可间接测定CEA. 由于标记的HRP可催化降解H2O2, 导致媒介体间苯二酚浓度改变, 使测定的灵敏度大大提高. 响应电流与CEA质量浓度的对数在2~160 ng/mL的范围内呈线性关系, 检出限为0.57 ng/mL(3σ法). 该免疫传感器具有制作简单、价廉及表面易于更新等特点.     

Ultrasensitive Eletrogenerated Chemiluminescence Immunoassay by Magnetic Nanobead Amplification

An ultrasensitive electrogenerated chemiluminescence (ECL) immunoassay was proposed by using magnetic nanobeads (MNBs) as the carrier of ECL labels for ECL emission amplification. Carcinoembryonic antigen (CEA) and MNBs were initially immobilized on a platform in 1 : 1 molar ratio via sandwich immunoreaction. Subsequently, the MNBs were released from the platform and labeled with Ru(bpy)₃²⁺ species. After the MNBs with Ru(bpy)₃²⁺ were immobilized on an Au electrode, ECL of the Ru(bpy)₃²⁺ was measured for CEA determination. A linear relation between the ECL intensity and CEA concentration was obtained in a range of 1×10^?14 to 3×10^?13 mol/L (2.0 to 60 pg/mL) with a limit of detection of 8.0×10^?15 mol/L (1.6 pg/mL).     

Electrochemiluminescence biosensor for carcinoembryonic antigen detection based on Au-Ag/g-C3N4 nanocomposites

Herein, an electrochemiluminescence (ECL) aptasensor for carcinoembryonic antigen (CEA) detection was developed based on Au-Ag/g-C₃N₄ nanocomposites (NCs), which were synthesized by decorating graphitic carbon nitride (g-C₃N₄) nanosheets with alloy-structured Au-Ag bimetallic nanoparticles (NPs) via one-step in situ chemical reduction. As ECL sensing platform, Au-Ag/g-C₃N₄ NCs could significantly improve the ECL intensity of luminol due to the good conductivity of Au-Ag NPs, electrocatalytic activity for oxygen evolution reaction (OER) and the ability to adsorb luminol via π stacking interaction. In addition, it could load the thiol terminated aptamers of CEA via Au-S or Ag-S bonds. In the presence of CEA, the ECL response of the proposed biosensor decreased significantly due to the fact that the assembled protein layers hindered the electron transfer and the diffusion of ECL reactants toward the electrode surface. The proposed ECL sensor exhibited a good linear relationship with CEA in the range of 1.0–1.0 × 10^?6 ng/mL with a detection limit of 8.9 × 10^?7 ng/mL. The satisfactory results were obtained in the detection of CEA in human serum samples.     

An electrochemical immunosensor for carcinoembryonic antigen enhanced by self-assembled nanogold coatings on magnetic particles

A quick and reproducible electrochemical-based immunosensor technique, using magnetic core/shell particles that are coated with self-assembled multilayer of nanogold, has been developed. Magnetic particles that are structured from Au/Fe₃O₄ core-shells were prepared and aminated after a reaction between gold and thiourea, and additional multilayered coatings of gold nanoparticles were assembled on the surface of the core/shell particles. The carcinoembryonic antibody (anti-CEA) was immobilized on the modified magnetic particles, which were then attached on the surface of solid paraffin carbon paste electrode (SPCE) by an external magnetic field. This is an assembly of a novel immuno biosensor for carcinoembryonic antigen (CEA). The sensitivity and response features of this immunoassay are significantly affected by the surface area and the biological compatibility of the multilayered nanogold. The linear range for the detection of CEA was from 0.005 to 50 ng mL⁻¹ and the limit of detection (LOD) was 0.001 ng mL⁻¹. The LOD is approximately 500 times more sensitive than that of the traditional enzyme-linked immunosorbent assay for CEA detection.     

Carbon Nanomaterials‐based Electrochemical Immunoassay with β‐Galactosidase as Labels for Carcinoembryonic Antigen

In this study, a novel signal‐amplified strategy for sensitive electrochemical sandwiched immunoassay of carcinoembryonic antigen (CEA) was constructed based on aminofunctionalized graphene oxide (GO‐NH₂) supported AgNPs used as catalytic labels of secondary anti‐CEA and β‐galactosidase (β‐Gal), Meanwhile, sulfhydrylation single‐wall carbon nanotubes (SWCNTs‐SH) as substrate materials embellished gold electrode through Au‐SH and connected with gold nanoparticles to form anti‐CEA/AuNPs/SWCNTs‐SH/Au sensing platform through layer‐by‐layer. In the presence of analyte CEA, a sandwich‐type immunoassay format was employed for determination of CEA by using the labeled β‐Gal toward the reduction of p‐aminophenyl galactopyranoside (PAPG) and the redox reaction of AgNPs. Under optimal conditions, the increase in the current was proportional to the concentration of CEA from 0.1 pg/mL to 200 ng/mL. The detection limit (LOD) was 0.036 pg/mL CEA at 3σ. The electrochemical immunoassay displayed an acceptable precision, selectivity, stability. Clinical serum specimens were assayed with the method, and the results were in acceptable agreement with those obtained from the referenced electrochemiluminescent method.     

A new dual immunoassay for tumor markers based on chemiluminescence signal amplification by magnetic mesoporous silica and enzyme modified gold nanoparticles

A sensitive dual immunoassay was proposed for the determination of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) based on signal amplification. Monoclonal antibodies immobilized on magnetic mesoporous silica particles (Fe(3)O(4)/SiO(2)) were prepared as the primary probe. Horseradish peroxidase (HRP) labeled antibodies co-coated with HRP on gold nanoparticles (AuNPs) were used as the secondary probe to achieve signal amplification. HRP tags were retained in the flow cells after a sandwich immunoassay. By controlling two switches on the two channels, chemiluminescent substrates were injected orderly man way, and then signals for CEA and AFP were sequentially detected by HRP-luminol-H(2)O(2). Due to the increased amount of HRP on AuNPs and the increased amount of monoclonal antibodies on Fe(3)O(4)/SiO(2), the signals were largely amplified. Under the optimal conditions, CEA and AFP could be detected in the linear ranges of 1.0 - 80 and 1.0 - 75 ng mL(-1) with detection limits of 0.25 and 0.5 ng mL(-1), respectively.     

One‐Step Electrochemical Immunoassay for Carcinoembryonic Antigen in Human via Back‐Filling Immobilization of Gold Nanoparticles on DNA‐Modified Gold Electrodes

A new amperometric immunobiosensor for carcinoembryonic antigen (CEA) determination in human serum was developed via encapsulation of horseradish peroxidase‐labeled carcinoembryonic antibody (HRP‐anti‐CEA) in a gold nanoparticles/DNA composite architecture. The presences of gold nanoparticles provided a congenial microenvironment for the immobilized biomolecules and decreased the electron transfer impedance, leading to a direct electrochemical behavior of the immobilized HRP. The formation of the antibody–antigen complex by a simple one‐step immunoreaction between the immobilized HRP‐anti‐CEA and CEA in sample solution introduced a barrier of direct electrical communication between the immobilized HRP and the gold electrode surface. Under optimal conditions, the current change obtained from the labeled HRP relative to H₂O₂ system was proportional to the CEA concentration in two linear ranges from 0.5 to 15 ng/mL and 15 to 300 ng/mL with a detection limit of 0.1 ng/mL (at 3δ). The precision and reproducibility are acceptable with the intraassay CV of 6.3% and 4.7% at 8 and 60 ng/mL CEA, respectively. The storage stability of the proposed immunosensor is acceptable in a pH 7.0 PBS at 4 °C for 9 days. Moreover, the proposed immunosensors were used to analyze CEA in human serum specimens. Analytical results of clinical samples show the developed immunoassay has a promising alternative approach for detecting CEA in the clinical diagnosis.     

Amperometric sandwich-type aptasensor based on peroxidase mimetic Au@Pt for the carcinoembryonic antigen

For sensitive analysis of cancer biomarker carcinoembryonic antigen (CEA), an amperometric sandwich-type aptasensor is proposed based on a signal amplification strategy of Au@Pt bimetallic nanoprobes. As the excellent catalytic activity to hydrogen peroxide (H₂O₂), core-shell Au@Pt nanoparticles are employed as nanoprobes by conjugating directly with the secondary aptamer of CEA (Apt-II). Due to the synergic recognition effect of dual aptamers and the excellent catalytic activity of nanoprobes, this amperometric sandwich-type aptasensor for CEA exhibits high specificity and good sensitivity with a limit of detection of 0.31 ng/mL, along with a wide linear range from 0.1 ng/mL to 100 ng/mL.     

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.     

Portable smartphone-based microfluidic electrochemical immunosensor for ultrasensitive detection of alpha-fetoprotein in human serum

Rapid and accurate tracing of biomarkers is essential for early detecting and diagnosing of cancer. Therefore, a valid and convenient strategy needs to be developed for efficient monitoring of cancer biomarkers. Herein, we constructed a portable microfluidic electrochemical immunosensor based on three-dimensional reduced graphene oxide (3D rGO) doped with gold nanoparticles (Au NPs) for ultrasensitive determination of alpha-fetoprotein (AFP). The designed microfluidic chip, with the advantages of small injection volume, detachable structure and high integration, was fabricated by 3D printing, which only needed 9 μL of reagent to realize the high sensitivity detection. In addition, the 3D Au NPs-rGO composites with high specific surface area and electrons transfer capacity can effectively increase electroactive sites and enhance electrochemical signals. Benefiting from these features, the 3D Au NPs-rGO microfluidic electrochemical immunochip showed a wide detection range between 0.1 pg/mL–200 ng/mL and a best detection limit of 0.045 pg/mL with the high sensitivity of 175.008 μA (ng/mL)⁻¹ cm⁻². Meanwhile, the proposed immunosensor exhibited reliable AFP detection in human serum samples, which demonstrated that this portable smartphone-based microfluidic electrochemical immunosensor hold great promises in clinical detection and huge potential in personalized healthcare.     

Electrochemical Immunoanalysis for Carcinoembryonic Antigen Based on Multilayer Architectures of Gold Nanoparticles and Polycation Biomimetic Interface on Glassy Carbon Electrode

This paper describes a layer‐by‐layer (LBL) self‐assembly process of chitosan (CTS) and gold nanoparticles (Au) on the pretreated negatively charged glassy carbon (GC) electrode to fabricate electrochemistry immunosensor with a nontoxic biomimetic interface, which provided an environment similar to a native system and allowed more freedom in orientation for immobilization of carcinoembryonic antibody (anti‐CEA) to monitor carcinoembryonic antigen (CEA). UV‐vis spectroscope, atomic force microscopy (AFM), and cyclic voltammetric (CV) measurements were used to follow the multilayer film formation. The performance of the biominetic interface and factors influencing the assay system were investigated in detail. The differential pulse voltammetry (DPV) current response is used for the CEA concentration assay. The dynamic range was from 0.50 to 80.00 ng mL^?1 with a detection limit of 0.27 ng mL^?1 at 3σ. In addition, the experiment results indicate that immobilization described in this proposed method exhibits a good sensitivity, selectivity, and stability.     

Determination of ultramicroquantities of Au(III) by its catalytic effect on 4-hydroxyl coumarone oxidation with potassium permanganate

A simple and fast kinetic method is based on the oxidation of 4-OH coumarone using KMnO₄ and the determination of ultramicroamounts of Au(III) by its catalytic effect on this reaction. The sensitivity of the method is 25 ng/mL. The relative error ranges between 9.20–3.90% for the concentration interval 5 × 10⁻⁸–2 × 10⁻⁷ g/mL. The selectivity of the method is very good, and the effect of foreign ions is investigated. The proposed approach has been applied to the determination of traces of Au(III) in copper ore. The text was submitted by the authors in English.     

Helical Substituted Polyacetylene‐Derived Fluorescent Microparticles Prepared by Precipitation Polymerization

This article reports the first fluorescent microparticles (MPs, approximately 600 nm in diameter) constructed using helical substituted polyacetylene and prepared via a precipitation polymerization approach. The MPs judiciously combine this interesting helical conjugated acetylene, fluorescent material and polymeric particles in one entity. The monomer containing a dansyl group undergoes precipitation polymerization in butanone/n‐heptane mixed solvent, with (nbd)Rh⁺B⁻(C₆H₅)₄ as a catalyst. MPs with a regular morphology are formed in a high yield (>80 wt%). UV‐vis spectroscopy demonstrates that the polymer chains making up the MPs adopt helical structures. The MPs show considerable fluorescence emission (λ_max, 500 nm; excited at 340 nm). Based on SEM and fluorescence images, the formation mechanism of the MPs is proposed. This methodology opens up new ways to prepare functional microstructured materials derived from substituted polyacetylenes, and may also result in opportunities for new practical applications of polyacetylene and its derivatives.

     

A simple double-bead sandwich assay for protein detection in serum using UV-vis spectroscopy

In this study a double-bead sandwich assay, employing magnetic nanoparticles and gold nanoparticles is proposed. The magnetic nanoparticles allow specific capturing of the analyte in biological samples, while the optical properties of the gold nanoparticles provide the signal transduction. We demonstrated that a major improvement in the assay sensitivity was obtained by selecting an optimal gold nanoparticle size (60 nm). A detection limit of 5-8 ng/mL, a sensitivity of 0.6-0.8 (pg/mL)⁻¹ and a dynamic range of 3 orders of magnitude were achieved without any further amplification using the detection of prostate specific antigen in serum as a model system. The proposed assay has the ability to be easily implemented within a microfluidic device for point-of-care applications whereby the readout can be executed by a fast and cheap optical measurement.     

Determination of Background Concentrations of Ag,Pd, Pt and Au in Highly Mineralized Ground Waters at Sub-ng L–1 Concentrations by Online Matrix Separation/Pre-Concentration Coupled to ICP-SFMS

Though not regulated in directives such as the Water Framework Directive of the European Union, the investigation of geogenic background concentrations of certain elements such as precious metals is of increasing interest, in particular for the early detection of a potential environmental pollution due to the increased use in various industrial and technological applications and in medicine. However, the precise and accurate quantification of precious metals in natural waters is challenging due to the complex matrices and the ultra-low concentrations in the (sub-) ng L⁻¹ range. A methodological approach, based on matrix separation and pre-concentration on the strong anion exchange resin TEVA^® Resin in an online mode directly coupled to ICP-SFMS, has been developed for the determination of Ag, Pt, Pd and Au in ground water. Membrane desolvation sample introduction was used to reduce oxide-based spectral interferences, which complicate the quantification of these metals with high accuracy. To overcome errors arising from matrix effects—in particular, the highly varying major ion composition of the investigated ground water samples—an isotope dilution analysis and quantification based on standard additions, respectively, were performed. The method allowed to process four samples per hour in a fully automated mode. With a sample volume of only 8 mL, enrichment factors of 6–9 could be achieved, yielding detection limits <1 ng L⁻¹. Validation of the trueness was performed based on the reference samples. This method has been used for the analysis of the total concentrations of Ag, Pt, Pd and Au in highly mineralized ground waters collected from springs located in important geological fault zones of Austria’s territory. Concentrations ranges of 0.21–64.2 ng L⁻¹ for Ag, 0.65–6.26 ng L⁻¹ for Pd, 0.07–1.55 ng L⁻¹ for Pt and 0.26–1.95 ng L⁻¹ for Au were found.     

Amperometric sensor for hydrogen peroxide based on direct electron transfer of spinach ferredoxin on Au electrode

A protein-based electrochemical sensor for hydrogen peroxide (H₂O₂) was developed by an easy and effective film fabrication method where spinach ferredoxin (Fdx) containing [2Fe–2S] metal center was cross linked with 11-mercaptoundecanoic acid (MUA) on a gold (Au) surface. The surface morphology of Fdx molecules on Au electrodes was investigated by atomic force microscopy (AFM). Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were employed to study the electrochemical behavior of adsorbed Fdx on Au. The interfacial properties of the modified electrode were evaluated in the presence of Fe(CN)₆^3?/4? redox couple as a probe. From CV, a pair of well-defined and quasi-reversible redox peaks of Fdx was obtained in 10 mM, pH 7.0 Tris–HCl buffer solution at ?170 and ?120 mV respectively. One electron reduction of the [2Fe-2S]²⁺ cluster occurs at one of the iron atoms to give the reduced [2Fe-2S]⁺. The formal reduction potential of Fdx ca. ?150 mV (vs. Ag/AgCl electrode) at pH 7.0. The electron-transfer rate constant, k_s, for electron transfer between the Au electrode and Fdx was estimated to be 0.12 s^?1. From the electrochemical experiments, it is observed that Fdx/MUA/Au promoted direct electron transfer between Fdx and electrode and it catalyzes the reduction of H₂O₂. The Fdx/MUA/Au electrode displays a linear increase in amperometric current for increasing concentration of H₂O₂.The sensor calibration plot was linear with r² = 0.998 with sensitivity approximately 68.24 μAm M^?1 cm^?2. Further, the effect of nitrite on the developed sensor was examined which does not interfere with the detection of H₂O₂. Finally, the addition of H₂O₂ on MUA/Au electrode was observed which has no effect on amperometric current.     

Ultrasensitive electrochemical immunosensor of carcinoembryonic antigen based on gold-label silver-stain signal amplification

A novel gold-label silver-stain electrochemical immunosensor based on polythionine-gold nanoparticles (PTh-Au NPs) modified glassy carbon electrode (GCE) as a platform and secondary antibody labeled Au NPs (Ab₂-Au NPs) as immumoprobe for carcinoembryonic antigen (CEA) detection. The sandwich-type biosensor adopted anodic stripping voltammetry to detect silver stripping signal when the Ab₂-Au NPs of the formed immunocomplexes were stained with silver.     

Evaluation of an Element-Tagged Duplex Immunoassay Coupled with Inductively Coupled Plasma Mass Spectrometry Detection: A Further Study for the Application of the New Assay in Clinical Laboratory

Background: Element-tagged immunoassay coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection has the potential to revolutionize immunoassay analysis for multiplex detection. However, a further study referring to the standard evaluation and clinical sample verification is needed to ensure its reliability for simultaneous analysis in clinical laboratories. Methods: Carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) were chosen for the duplex immunoassay. The performance of the assay was evaluated according to guidelines from the Clinical and Laboratory Standards Institute (CLSI). Moreover, reference intervals (RIs) of CEA and AFP were established. At last, 329 clinical samples were analyzed by the proposed method and results were compared with those obtained with electrochemiluminescent immunoassay (ECLIA) method. Results: The measurement range of the assay was 2–940 ng/mL for CEA and 1.5–1000 ng/mL for AFP, with a detection limit of 0.94 ng/mL and 0.34 ng/mL, respectively. The inter-assay and intra-assay imprecision were all less than 6.58% and 10.62%, respectively. The RI of CEA and AFP was 0–3.84 ng/mL and 0–9.94 ng/mL, respectively. Regarding to clinical sample detection, no significant difference was observed between the proposed duplex assay and the ECLIA method. Conclusions: The ICP-MS-based duplex immunoassay was successfully developed and the analytical performance fully proved clinical applicability. Well, this could be different with other analytes.