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.     

Interdigitated Conductometric Immunosensor for Determination of Interleukin‐6 in Humans Based on Dendrimer G4 and Colloidal Gold Modified Composite Film

A highly sensitive, fast and stable conductometric immunosensor for determination of interleukin‐6 (IL6) in humans is developed by encapsulation of horseradish peroxidase‐labeled interleukin‐6 antibody (HRP‐anti‐IL6) in poly(amidoamine) fourth‐generation dendrimer (dendrimer) and colloidal gold (nanogold) modified composite architecture. The presences of nanogold and dendrimer 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‐IL6 and IL6 in sample solution introduced a barrier of direct electrical communication between the immobilized HRP and the gold electrode surface, thus local conductivity variations could be detected by the HRP electrocatalytic reaction in 0.02 M phosphate buffer solution (pH 7.0) containing 50 μM H₂O₂, 0.01 M KI and 0.15 M NaC1. Under optimal conditions, the proposed immunosensor exhibited a good conductometric response to IL6 in a linear range from 30 to 300 pg/mL with a relatively low detection limit of 10 pg/mL at 3δ. The precision and reproducibility are acceptable with the intra‐assay CV of 7.3% and 5.6% at 100 and 200 pg/mL IL6, respectively. The storage stability of the proposed immunosensor is acceptable in a pH 7.0 PBS at 4 °C for 8 days. Importantly, the proposed methodology could be extended to the detection of other antigens or biocompounds.     

Electroanalytical Determination of Carcinoembryonic Antigen at a Silica Nanoparticles/Titania Sol–Gel Composite Membrane‐Modified Gold Electrode

A highly hydrophilic and nontoxic colloidal silica nanoparticle/titania sol–gel composite membrane was prepared on a gold electrode via a chemical vapor deposition method. With carcinoembryonic antigen (CEA) as a model antigen and encapsulation of carcinoembryonic antibody (anti‐CEA) in the composite architecture, this membrane could be used for reagentless electrochemical immunoassay. The presence of silica nanoparticles provided a congenial microenvironment for adsorbed biomolecules. The formation of immunoconjugate by a simple one‐step immunoreaction between CEA in sample solution and the immobilized anti‐CEA introduced the change in the potential. The modified procedure was further characterized by electrochemical impedance spectroscopy and cyclic voltammetry. Compared to the commonly applied methods, i.e., the TiO₂ direct embedding procedure, this strategy could allow for antibodies immobilized with higher loading amount and better retained immunoactivity. The resulting immunosensor exhibited high sensitivity, good precision, acceptable stability, accuracy, reproducibility and wide linear range from 1.5 to 240 ng mL^?1 with a detection limit of 0.5 ng mL^?1 at 3σ. Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme‐linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting CEA in the clinical diagnosis. Furthermore, this composite membrane could be used efficiently for the entrapment of other biomarkers and clinical applications.     

Dual‐Amplification of Antigen–Antibody Interactions via Backfilling Gold Nanoparticles on (3‐Mercaptopropyl) Trimethoxysilane Sol‐Gel Functionalized Interface

A new dual‐amplification strategy of electrochemical signaling from antigen–antibody interactions was proposed via backfilling gold nanoparticles on (3‐mercaptopropyl) trimethoxysilane sol‐gel (MPTS) functionalized interface. The MPTS was employed not only as a building block for the electrode surface modification but also as a matrix for ligand functionalization with first amplification. The second signal amplification strategy introduced in this study was based on the backfilling immobilization of nanogold particles to the immunosensor surface. Several coupling techniques, such as with nanogold but not MPTS or with MPTS but not nanogold, were investigated for the determination of carcinoembryonic antigen (CEA) as a model, and a very good result was obtained with nanogold and MPTS coupling immunosensor. With the noncompetitive format, the formation of the antigen–antibody complex by a simple one‐step immunoreaction between the immobilized anti‐CEA and CEA in sample solution introduced membrane potential change before and after the antigen–antibody interaction. Under optimal conditions, the proposed immunosensor exhibited a good electrochemical behavior to CEA in a dynamic concentration range of 4.4 to 85.7 ng/mL with a detection limit of 1.2 ng/mL (at 3 δ). Moreover, the precision, reproducibility and stability of the as‐prepared immunosensor were acceptable. Importantly, the proposed methodology would be valuable for diagnosis and monitoring of carcinoma and its metastasis.     

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.     

Gold Nanoparticles Doped Three‐Dimensional Sol‐gel Matrix for Amperometric Human Chorionic Gonadotrophin Immunosensor

A novel electrochemical immunosensor for human chorionic gonadotrophin (hCG) was proposed by immobilization of hCG in gold nanoparticles doped three‐dimensional (3D) sol‐gel matrix and an interfacial competitive immunoreaction. The 3D organized composite structure was prepared by assemble of gold nanoparticles into a hydrolyzed (3‐mercaptopropyl)‐trimethoxysilane sol‐gel matrix, which showed good biocompatibility. After the interfacial competitive immunoreaction the formed HRP‐labeled immunoconjugate showed good enzymatic activity for the oxidation of o‐phenylenediamine by H₂O₂. With a competitive format, a method comprising of o‐phenylenediamine‐H₂O₂‐immobilized HRP labeled hCG immunoconjugate system for immunoassay of hCG from 5.0 to 30.0 mIU mL^?1 was developed. The immunosensor showed good precision, high sensitivity, acceptable stability and reproducibility and could be used for detection of hCG in human serum with the consistent results in comparison with those obtained by a commercial analyzer.     

Biomolecule‐Doped Organic/Inorganic Hybrid Nanocomposite Film for Label‐Free Electrochemical Immunoassay of α‐1‐Fetoprotein

A new electrochemical immunosensor for the detection of α‐1‐fetoprotien (AFP) was developed based on AFP antibody (anti‐AFP)‐functionalized organic/inorganic hybrid nanocomposite membrane. To fabricate such a hybrid composite membrane, 3,4,9,10‐perylenetetracarboxylic acid‐bound thionine molecules (PTCTH) were initially doped into titania colloids (TiO₂), and then gold nanoparticles and anti‐AFP were immobilized onto the composite film in turn. Comparison with the electrode fabricated only with thionine not 3,4,9,10‐perylenetetracarboxylic acid, the immunosensor with PTCTH exhibited high sensitivity and fast electron transfer. The presence of gold nanoparticles provided a good microenvironment for the immobilization of biomolecules, enhanced the surface coverage of protein, and improved the sensitivity of the immunosensor. The modified process was characterized by scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The surface topography of the membrane was investigated by scanning electron microscopy (SEM). Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 2.5 to 200.0 ng/mL towards AFP with a detection limit of 0.5 ng/mL (S/N=3). The stability, reproducibility and precision of the immunosensor were acceptable. Comparison with the conventional enzyme‐linked immunosorbent assay (ELISA), the present method did not require more labeled procedures and washing steps. Significantly, the detection methodology provides a promising approach for other proteins or biosecurities.     

Electrochemical Immunoassay of Human Chorionic Gonadotrophin Based on Its Immobilization in Gold Nanoparticles‐Chitosan Membrane

A human chorionic gonadotrophin (hCG) doped gold nanoparticles–chitosan membrane was prepared for forming an immunoconjugate of horseradish peroxidase labeled hCG antibody and hCG on glassy carbon electrode. The nanoparticles provided a congenial environment of the adsorbed proteins. Thus, the immobilized HRP‐labeled immunoconjugate showed good enzymatic activity for the oxidation of o‐phenylenediamine by H₂O₂. With a competitive mechanism, an amperometric method for immunoassay of hCG up to 30 mIU mL^?1 with a relatively low detection limit of 0.26 mIU mL^?1 at 3σ was developed. The hCG immunosensor showed good precision, high sensitivity, acceptable stability and reproducibility.     

Ultrasensitive Electrochemical Immunosensor for Detection of Tumor Necrosis Factor‐α Based on Functionalized MWCNT‐Gold Nanoparticle/Ionic Liquid Nanocomposite

A novel and highly sensitive electrochemical immunosensor was developed for the detection of protein biomarker tumor necrosis factor‐alpha (TNF‐α) based on immobilization of TNF‐α‐antibody (anti‐TNF‐α) onto robust nanocomposite containing gold nanoparticles (AuNP), multiwalled carbon nanotubes (MWCNTs) and ionic liquid (1‐buthyl‐3‐methylimidazolium bis (trifluoromethyl sulfonyl)imide). Functionalized MWCNT‐gold nanoparticle was produced by one‐step synthesis based on the direct redox reaction. The electrochemical properties of nanocomposite were characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The anti‐TNF‐α was immobilized or entrapped in the nanocomposite and used in a sandwich type complex immunoassay with anti‐TNF‐α labeled with horseradish peroxidase as secondary antibody. Under optimum conditions, the immunosensor could detect TNF‐α in a linear range from 6.0 to 100 pg mL^?1 with a low detection limit of 2.0 pg mL^?1. The simple fabrication method, high sensitivity, good reproducibility, stability, as well as acceptable accuracy for TNF‐α detection in human serum samples are the main advantages of this immunosensor, which might have broad applications in protein diagnostics and bioassay.     

一种快速高选择性检测痕量癌胚抗原的共振散射光谱法

用10 nm的金纳米粒子标记单克隆癌胚抗原抗体制备了检测癌胚抗原（CEA）的共振散射光谱探针（Au-CEAAb）。在pH 6.8 的Na2HPO4- NaH2PO4缓冲溶液中及聚乙二醇-6000存在下, CEA与Au-CEAAb发生免疫反应聚集形成疏水性的、平均粒径为227.0 nm的免疫复合物微粒,并在321 nm、581 nm产生2个共振散射峰。随着癌胚抗原（CEA）浓度的增大,581 nm处的共振散射强度I581nm线性增加,其增加值△I581nm与CEA浓度在1.0～50.0 ng·mL-1范围内呈良好的线性关系,相应的回归方程、相关系数、检出限(3σ)分别为ΔI581nm=1.63 C +5.6、0.9940、0.52 ng·mL-1。该法简便、快速、灵敏且选择性好,用于检测人血清中癌胚抗原（CEA）,结果满意。     

Electrochemical immunosensor for carcinoembryonic antigen based on antigen immobilization in gold nanoparticles modified chitosan membrane.

A disposable electrochemical immunosensor for carcinoembryonic antigen (CEA) was proposed based on the antigen immobilized in a colloidal gold nanoparticles modified chitosan membrane on the surface of an indium-tin oxide (ITO) electrode. The different membranes were characterized by scanning electron microscope and electrochemical methods. Based on a competitive immunoassay format, the immobilized antigen of the immunosensor was incubated with a horseradish peroxidase (HRP) labeled antibody and sample CEA antigen, and the formed immunoconjugate in the immunosensor was detected by an o-phenylenediamine-H(2)O(2)-HRP electrochemical system. Under the optimal experimental conditions, the electrocatalytic current decreased linearly with the competitive mechanism. CEA could be determined in the linear range from 2.0 to 20 ng/ml with a detection limit of 1.0 ng/ml. The prepared CEA immunosensor is not only economic due to the low-cost ITO electrode obtained from industrial mass production, but is also capable with good stability and reproducibility for batch fabrication.     

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.     

基于纳米金与碳纳米管-纳米铂-壳聚糖纳米复合物固定癌胚抗原免疫传感器的研究

采用纳米金(nano-Au)、多壁碳纳米管-纳米铂-壳聚糖的纳米复合物(MWNT-Pt-CS)及电子媒介体硫堇(Th)固载抗体制得高灵敏癌胚抗原免疫传感器．首先, 于壳聚糖溶液中用NaBH4还原H2PtCl6, 并将多壁碳纳米管分散于其中制得碳纳米管-纳米铂-壳聚糖纳米复合物, 并将其滴涂在玻碳电极上成膜; 然后, 吸附电子媒介体硫堇制得硫堇/碳纳米管-纳米铂-壳聚糖(Th/MWNT-Pt-CS)修饰电极．利用壳聚糖和硫堇分子中大量的氨基固定纳米金并吸附癌胚抗体(anti-CEA); 最后, 用辣根过氧化物酶(HRP)封闭活性位点从而制得高灵敏电流型免疫传感器．在优化的实验条件下, 该传感器响应的峰电流值与癌胚抗原(carcinoembryonic antigen)浓度在0.5～10和10～120 ng/mL的范围内保持良好的线性关系, 检测限为0.2 ng/mL．     

A Reagentless Amperometric Immunosensor for Alpha‐Fetoprotein Based on Gold Nanoparticles/TiO2 Colloids/Prussian Blue Modified Platinum Electrode

A novel reagentless amperometric immunosensor for the determination of alpha‐fetoprotein (AFP) was prepared by immobilizing TiO₂ colloids on Prussian blue (PB) modified platinum electrode, which yielded a positively charged interface with strong adsorption to deposit gold nanoparticles for immobilization of alpha‐fetoprotein antibody (anti‐AFP). The factors influencing the performance of the proposed immunosensors were studied in detail. Under the optimized conditions, cyclic voltammograms determination of AFP showed a specific response in two concentration ranges from 3.0 to 30.0 ng/mL and from 30.0 to 300.0 ng/mL with a detection limit of 1.0 ng/mL at a signal‐to‐noise ratio of 3. The proposed immunosensor exhibited high selectivity, good reproducibility, long‐term stability (>2 months) and good repeatability.     

Ultrasensitive electrochemiluminescence immunoassay for tumor marker detection using functionalized Ru-silica@nanoporous gold composite as labels

In this work, we reported a simple and sensitive sandwich-type electrochemiluminescence (ECL) immunosensor for carcinoembryonic antigen (CEA) on a gold nanoparticles (AuNPs) modified glassy carbon electrode (GCE). The Ru-silica (Ru(bpy)(3)(2+)-doped silica) capped nanoporous gold (NPG) (Ru-silica@NPG) composite was used as an excellent label with amplification techniques. The NPG was prepared with a simple dealloying strategy, by which silver was dissolved from silver/gold alloys in nitric acid. The primary antibody was immobilized on the AuNPs modified electrode through l-cysteine and glutaraldehyde, and then the antigen and the functionalized Ru-silica@NPG composite labeled secondary antibody were conjugated successively to form a sandwich-type immunocomplex through the specific interaction. The concentrations of CEA were obtained in the range from 1 pg mL(-1) to 10 ng mL(-1) with a detection limit of 0.8 pg mL(-1). The as-proposed ECL immunosensor has the advantages of high sensitivity, specificity and stability and could become a promising technique for tumor marker detection.     

基于酶标抗体和媒介体共吸附于金胶壳聚糖膜的PSA免疫传感器的制备

本文研制了一种用金胶壳聚糖仿生膜来同时固定四甲基联苯胺（TMB）和酶标抗体的新型电化学免疫传感器,用于检测血清肿瘤标志物前列腺特异性抗原（PSA）的含量。固定的TMB作为电子传递媒介体,在扫速小于45 mV/s时,电极表现为一个表面控制过程,而在扫速大于45 mV/s时则表现为一个扩散控制过程。将固定有酶标抗体和TMB的免疫传感器与待测PSA抗原一起培育,在该传感器上形成的免疫复合物通过TMB-H₂O₂-HRP电化学体系进行了测定。在优化实验条件下,PSA的线性检测范围为5-30 ng·mL^-1,检测限为1.0 ng·mL^-1。该PSA免疫传感器制备方法简单,成本低廉,具有较好的稳定性和重现性。     

Gold Nanoparticle‐Based Redox Signal Enhancement for Sensitive Detection of Human Chorionic Gonadotropin Hormone

A sensitive immunosensor for the detection of pregnancy marker, human chorionic gonadotropin hormone (hCG), was developed using the direct electrical detection of Au nanoparticles. We utilized disposable screen‐printed carbon strips (SPCSs) for the development of our immunosensor, which provided cost‐effective tests with the required antigen sample volume as small as 2 μL. After the recognition reaction between the surface‐immobilized primary antibody and hCG, the captured antigen was sandwiched with a secondary antibody that was labeled with Au nanoparticles. Au nanoparticles were exposed to a preoxidation process at 1.2 V for 40 s, which was subsequently followed with a reduction scan on the same surface using differential pulse voltammetry (DPV). We could observe Au nanoparticle‐labeled antigen‐antibody complexes immobilized on the surface of SPCS using scanning electron microscopy (SEM). Additionally, the number of Au nanoparticles on the immunosensor was determined using SEM images, and showed a linear relationship with the current intensity obtained from the DPV measurements with a detection limit of 36 pg/mL hCG (612 fM, 3.6×10^?4 IU/mL). Our immunosensor system, a combination of the screen‐printing technology with Au nanoparticles provides a promising biosensor for various applications in life sciences.     

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.     

A Renewable Potentiometric Immunosensor Based on Fe3O4 Nanoparticles Immobilized Anti‐IgG

A renewable potentiometric immunosensor for detection of immunoglobulin G (IgG) has been developed by magnetic force attraction of Fe₃O₄ nanoparticles immobilized goat‐anti‐human IgG antibody. For preparing sensitive film of the sensor, cysteine was bonded on the nano‐Fe₃O₄ particles surface. The cysteine functionalized magnetic nanoparticles was attracted on a solid paraffin carbon paste electrode surface to covalently immobilize of anti‐immunoglobulin G (anti‐IgG) by employing a conventional glutaraldehyde‐crosslinking method. The immunosensor showed a specific response to human immunoglobulin G in the range of 0.1–1.2 ng/mL with a detection limit of 0.023 ng/mL. The immunosensor based on the magnetic nanoparticles was made easily by this method. It can be used expediently, renewed easily and low‐cost relatively. The renewable potentiometric immunosensor with better stability and higher sensitivity can be employed extensively in clinical diagnosis, monitoring of disease and environmental studies and etc.     

An Amperometric Immunosensor Based on Layer‐by‐Layer Assembly of L‐Cysteine and Nanosized Prussian Blue on Gold Electrode for Determination of Human Chorionic Gonadotrophin

A novel amperometric immunosensor based on L ‐cysteine/nanosized Prussian blue bilayer films ({NPB/L ‐cys}₂) and gold nanoparticles (nano‐Au) was fabricated for determination of human chorionic gonadotrophin (HCG). First, L ‐cys and NPB was self‐assembled by layer‐by‐layer (LBL) technology to form {NPB/L ‐cys}₂ bilayer films on the gold electrode. Subsequently, nano‐Au layer was immobilized on the {NPB/L ‐cys}₂ bilayer films by electrodepositing gold chloride tetrahydrate and then anti‐HCG was assembly on the nano‐Au layer. Finally hemoglobin (Hb) was employed to block sites against nonspecific binding. With the electrocatalytic ability of Hb and NPB for the reduction of H₂O₂, the current signal of the antigen‐antibody reaction was amplified and the enhanced sensitivity was achieved. In this study, the assembly process and performance of the immunosensor were characterized by cyclic voltammetry (CV) and the morphology was researched by scanning electron microscopy (SEM). The immunosensor performed a high sensitivity and a wide linear response to HCG in two ranges from 0.5 to 10 mIU/mL and from 10 to 200 mIU/mL with a relatively low detection limit of 0.2 mIU/mL at 3 times the background noise, as well as good stability and long‐term life.