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.     

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.     

A novel piezoelectric immunosensor for detection of carcinoembryonic antigen

A simple, rapid, and highly sensitive immunosensor for the direct determination of carcinoembryonic antigen (CEA) in human serum using a piezoelectric crystal has been developed and optimized. In order to improve sensitivity of the immunosensor, a protein A-based orientation-controlled immobilization method for antibodies was adopted together with an immunoreactive accelerant of polyethyleneglycol (PEG) used to amplify the signal response of frequency. Human normal serum was utilized as a reference background. The linear range for CEA concentration obtained by the end-point method was 66.7-466.7 ng/mL. Clinical samples from cancer patients were analyzed by the proposed piezoelectric immunoassay, and the analytical results were reasonably comparable with those obtained by the chemiluminescence immunoassay (CLIA). The proposed immunosensor provides a new promising method for the highly sensitive immunoassay of CEA in clinical laboratory.     

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.     

Magnetic core-shell Fe3O4@Ag nanoparticles coated carbon paste interface for studies of carcinoembryonic antigen in clinical immunoassay

This study demonstrates a novel approach toward development of advanced immunosensors based on chemically functionalized core-shell Fe3O4@Ag magnetic nanoparticles, and the preparation, characterization, and measurement of relevant properties of the immunosensor useful for the detection of carcinoembryonic antigen (CEA) in clinical immunoassay. The immunosensor based on the combination of a magnetic nanocore and an Ag metallic shell shows good adsorption properties for the attachment of the CEA antibody selective to CEA. The core-shell nanostructure presents good magnetic properties to facilitate and modulate the way it was integrated into a carbon paste. Under optimal conditions, the resulting composite presents good electrochemical response for the detection of CEA, and allows detection of CEA at a concentration as low as 0.5 ng.mL(-1). Importantly, the proposed methodology could be extended to the detection of other antigens or biocompounds.     

Highly sensitive rapid chemiluminescent immunoassay using the DNAzyme label for signal amplification

A novel trace tag for chemiluminescent (CL) immunoassay was designed by using DNAzyme to functionalize antibody-labeled Au nanoparticles (AuNPs). The trace tag showed an excellent ability to catalyze the oxidation of luminol by hydrogen peroxide, leading to strong CL emission. By coupling the trace tag with a passive mixing accelerated immunoreaction system, a highly sensitive rapid flow-through CL immunoassay method was proposed. Using carcinoembryonic antigen (CEA) as a model analyte, the capture antibody for CEA was immobilized on paramagnetic microspheres, and DNAzyme-anti-CEA antibody functionalized AuNPs were prepared as trace tag. A three-dimensional helical glass tube kept at 37 °C in a water bath was used for passively mixing immunoreagents in a two-step sandwich immunoassay, with which each immunoreaction step could be finished within 150 s. With the help of a magnet, the immunocomplex could conveniently be separated from reactants. Compared with the horseradish peroxidase-based tag, the newly designed trace tag showed obvious signal amplification due to its strong catalytic ability and high loading ratio of DNAzyme on each AuNP. The proposed method showed a linear calibration range from 0.005 to 0.5 ng mL(-1) for CEA detection with a detection limit of 4.1 pg mL(-1) at a signal-to-noise ratio of 3 and acceptable detection reproducibility. The assay results of clinical serum samples were in acceptable agreement with the reference values. The designed immunoassay system with ultrahigh sensitivity provided a programmable and low-cost approach for high-throughput clinical application.     

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。     

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.     

3,3'-diaminobenzidine (DAB)-H2O2-HRP voltammetric enzyme-linked immunoassay for the detection of carcionembryonic antigen

A new voltammetric enzyme-linked immunoassay system of 3,3'-diaminobenzidine (DAB)-H2O2-horseradish peroxidase (HRP) has been presented and used for the sensitive detection of carcinoembryonic antigen (CEA) in human serum. In this proposed procedure, DAB was firstly used as the electroactive substrate in the HRP catalyzed oxidation reaction in the present of H2O2. The generated product produced a sensitive second-order derivative linear sweep voltammetric peak at potential of -0.62 V (vs. SCE) in Britton-Robinson (BR) buffer solution. The free HRP could be measured in a linear range from 2.5 x 10(-6)-2.5 x 10(-2) unit/ml and a detection limit of about 1.5 x 10(-6) unit/ml. Under the optimal experiment conditions, CEA could be detected in the linear range from 0.50 to 80 ng/ml with a detection limit of 0.5 ng/ml. The proposed electrochemical enzyme-linked immunosorbent assay method is simple, inexpensive, reproducible and sensitive, which shows promising for detecting CEA in the clinical diagnosis.     

CE-based simultaneous liquid-phase noncompetitive enzyme immunoassay for three tumor markers in human serum using electrochemical detection

A method for indirectly detecting horseradish peroxidase (HRP) was described by CE with electrochemical detection. Details of selection for optimum conditions were presented. The detection limit of free HRP was 1.09 x 10(-12) M or 0.94 zmol (S/N = 3). A novel CE-based liquid-phase binding noncompetitive enzyme immunoassay (CE-EIA) was developed. In this method, after the noncompetitive immunoreaction in liquid phase, the free enzyme (HRP)-labeled antibody (Ab*) and the bound enzyme-labeled complex (Ag-Ab*) were separated and then the system of HRP catalyzing H(2)O(2)/o-aminophenol (OAP) reaction was adopted. Prostate specific antigen (PSA), carcinoembryonic antigen (CEA), and human chorionic gonadotropin (HCG) in human serum samples were detected without any sample preparation, with the detection limits (S/N = 3) of 0.22, 0.17 and 0.30 ng/mL, respectively. This technique has been successfully applied to detect simultaneously PSA, CEA, and HCG in 12 min, upon adding these three antigens into human serum to simulate patient serum. It proves that the CE-EIA technique proposed could be developed into a sensitive and new method for simultaneous clinical assay of multianalytes.     

Surface Plasmon Resonance Immunoassay for Ochratoxin A Based on Nanogold Hollow Balls with Dendritic Surface

Abstract

A surface plasmon resonance (SPR)‐immunosensor based on nano‐size gold hollow ball (GHB) with dendritic surface has been developed for detection of Ochratoxin A (OTA). A thionine thin film was initially electropolymerized onto the SPR‐probe surface, and then anti‐OTA monoclonal antibody (anti‐OTA) was immobilized onto the SPR‐probe surface by means of GHB conjugation. The binding of target molecules onto the immobilized antibodies causes an increase in the resonant angle of the sensor chip, and the resonant angle shift was proportional to the OTA concentration in the range of 0.05–7.5 ng/ml with a detection limit of 0.01 ng/ml at a signal/noise ration of 3. A glycine‐HCl solution (pH 2.8) was used to release antigen‐antibody complexes from the biorecognition surface. Good reusability was exhibited. Moreover, spiking various levels of OTA into three milk samples was assayed using the proposed immunoassay. Analytical results show the precision of the developed immunoassay is acceptable. Compared with the conventional enzyme‐linked immunosorbent assay, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.     

Development of a highly sensitive and specific immunoassay for determining chrysoidine, a banned dye, in soybean milk film

A highly specific and sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA）was developed for the first time for the detection of chrysoidine, a dye banned in soybean milk film. Two haptens with different spacer arms were synthesized to produce antibodies. Both homologous and heterologous immunoassay formats were compared to enhance the icELISA sensitivity. The heterologous icELISA exhibited better performance, with an IC(50) (50% inhibitory concentration) of 0.33 ng/mL, a limit of detection (LOD, 10% inhibitory concentration) of 0.04 ng/mL, and a limit of quantitation (LOQ, 20%-80% inhibitory concentration) from 0.09 to 4.9 ng/mL. The developed icELISA was high sensitive and specific, and was applied to determine chrysoidine in fortified soybean milk film samples. The results were in good agreement with that obtained by high-performance liquid chromatography (HPLC) analyses.     

Sensitive electrochemical immunosensor array for the simultaneous detection of multiple tumor markers

A novel electrochemical immunosensor array for the simultaneous detection of multiple tumor markers was developed by incorporating electrochemically addressing immobilization and one signal antibody strategy. As a proof-of-principle, an eight-electrode array including six carbon screen-printed working electrodes was used as a base array for the analysis of two important tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) and a horseradish peroxidase-labeled antibody was employed as a signal antibody. The immunosensor in the array was fabricated in sequence by covalently coupling the capture antibody onto the surface of the desired working electrode, which was firstly electrochemically addressably grafted with an aminophenyl group by reduction of in situ generated aminophenyl diazonium cation generated from p-phenylenediamine, using glutaraldehyde as cross-linker. This allowed the selective immobilization of the capture antibody at the desired position on a single array via an electrochemical operation. The immunoassay in sandwich mode was performed by specifically binding the targets, second antibodies and one signal antibody to the immunosensor array. The result showed that the steady current density was directly proportional to the concentration of target CEA/AFP in the range from 0.10 to 50 ng mL(-1) with a detection limit of 0.03 ng mL(-1) for CEA and 0.05 ng mL(-1) for AFP (S/N = 3), respectively. This work demonstrates that the employment of an electrochemically addressing method for the fabrication of an immunosensor array and one signal antibody is a promising approach for the determination of multiple tumor markers in clinical samples.     

Nitrogen-doped TiO2 Nanocrystals for Highly Sensitive Electrochemical Immunoassay of Carcinoembryonic Antigen

Nitrogen-doped TiO₂ nanocrystals (N−TiO₂ NCs) were simply synthesized and then functionalized with streptavidin for highly sensitive electrochemical immunoassay of tumor marker. Scanning electron microscopy, transmission electron microscopy, static water contact angle, and cyclic voltametric measurement were adapted to examine the properties of N−TiO₂ NCs and resultant immunosensor. The functionalized N−TiO₂ NCs sensing platform shows high electrochemical conductivity, large surface area and excellent hydrophilicity. The features make them to produce high current response, capture more antibody molecules, and maintain the bioactivity of immobilized antibodies. By means of carcinoembryonic antigen (CEA) as model tumor marker, a wide linear range of 0.005–3 ng/mL and a low detection limit of 0.005 ng/mL (signal-to-noise ratio of 3) were achieved by the proposed CEA immunosensor. Furthermore, the resultant CEA immunosensor displays high specificity and was employed to determine CEA in clinical serum samples.     

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．     

Simultaneous detection of two lung cancer biomarkers using dual-color fluorescence quantum dots

Quantum dots (QDs) have the potential to simplify the performance of multiplexed analysis. In this work, a novel protocol for performing a simultaneous dual-protein immunoassay, i.e. two lung cancer biomarkers, carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE), based on dual-color QDs, is described. First, two capture antibodies (both with biotin tags), two antigens and two detection antibodies were mixed together and the sandwich complexes were thus formed in the homogeneous solution, and then streptavidin coated polystyrene beads were directly added into the resultant system. Bead aggregation can be made self-limiting by controlling the shaker speed during the immunoassay. A distinct transition occurs between limited and complete aggregation as a function of the shaker speed during the immunoassay. Second, dual-color QDs with emission maxima at 525 and 655 nm were added after washing and reacted with the corresponding detection antibodies. Third, the bead-QD conjugates were dissociated in the dissociation buffer and then free QDs were directly used for the fluorescence detection of CEA and NSE. The results show that CEA and NSE could be sensitively determined with a common 96-well fluorescence plate reader and with equal detection limits down to the 1.0 ng mL(-1) level. Within the calibrated amount, the protocol had excellent precision within 0.53% for each target and was comparable in performance to commercial single-analyte ELISAs. Furthermore, the proposed method has been successfully applied to the determination of dual markers in real samples without cross-reaction, and a good correlation was achieved after comparison with the conventional assay for CEA and NSE in 25 human serum samples.     

Biofunctionalized dendritic polyaniline nanofibers for sensitive electrochemical immunoassay of biomarkers

Multi-armed dendritic polyaniline nanofibers (MPANFs) were first synthesized and functionalized with horseradish peroxidase (HRP) and carcinoembryonic antibody (anti-CEA) for highly efficient electrochemical immunoassay of carcinoembryonic antigen (CEA, as a model analyte here) in this work. Transmission electron microscope (TEM) and scanning electron microscope (SEM) techniques were employed to characterize the synthesized MPANFs. By using anti-CEA-conjugated core-shell gold-Fe(3)O(4) nanocomposites (GoldMag) as immunosensing probes and biofunctionalized MPANFs as molecular tags, a new sandwich-type homogeneous immunoassay strategy was developed for the determination of CEA by coupling with a home-made flow-through magneto-controlled microfluidic device. Under optimal conditions, the electrochemical immunoassay exhibited a wide dynamic range of four orders of magnitude from 1.0 pg mL(-1) to 50 ng mL(-1) with a low detection limit of 0.1 pg mL(-1) CEA at 3σ. Intra- and inter-assay coefficients of variation were below 10%. The assayed results for clinical serum specimens with the electrochemical immunoassay were received in good accordance with the results obtained from the referenced enzyme-linked immunosorbent assay (ELISA) method.     

Automated chemiluminescent dual-analyte immunoassay based on resolved immunosensing channels

A novel system of series-wound immunosensing channels (SWIC) was proposed for automated chemiluminescent (CL) dual-analyte immunoassay by immobilizing respectively different capture antibodies on the inner walls of series-wound glass channels. This system could use a single enzyme as label to perform multiplex immunoassay in one fluid way. Using α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as model analytes, the mixture including AFP, horseradish peroxidase (HRP)-labeled anti-AFP antibody, CEA and HRP-labeled anti-CEA antibody was introduced into the SWIC for carrying out the on-line incubation. Upon injection of CL substrate the CL signals from the two immunosensing channels were conveniently resolved and near-simultaneously collected with the aid of optical shutter. AFP and CEA could be rapidly assayed in the ranges of 1.0-100 and 1.0-80 ng/ml with detection limits of 0.41 and 0.39 ng/ml, respectively. The assay results of clinical serum samples were in an acceptable agreement with the reference values. This designed flow-through immunosensing system based on SWIC provided an automated, reusable, simple, sensitive and low-cost approach for multianalyte immunoassay.     

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.     

A reusable electrochemical immunosensor for carcinoembryonic antigen via molecular recognition of glycoprotein antibody by phenylboronic acid self-assembly layer on gold

A reusable amperometric immunosensor based on the reversible boronic acid-sugar interaction is proposed. The immunosensor was prepared by self-assembling a thiol-mixed monolayer comprised of conjugates of 3-aminophenylboronic acid with 11-mercaptoundecanoic acid (APBA-MUA) and 11-mercapto-1-undecanol (MU) on gold. The resulting boronic acid coating layer can specifically bind with the glycoprotein antibody, enzyme conjugated carcinoembryonic antibody (HRP-anti-CEA). Voltammetric and electrochemical impedance spectroscopic (EIS) studies and surface plasmon resonance (SPR) measurements show that the binding of HRP-anti-CEA to the APBA interface is reversible and the HRP-anti-CEA can be removed with an acidic buffer or a solution containing sorbitol. The bound enzyme-conjugated antibody can retain its enzyme catalytic activity to the reduction of hydrogen peroxide (H(2)O(2)) and its immunoactivity while binding with CEA to form an immunocomplex. After the formation of the immunocomplex, the access of the active center of HRP to thionine was partially inhibited. This leads to a linear decrease in the electrocatalytic response of HRP-anti-CEA-modified electrode over a CEA concentration range of 2.5 to 40.0 ng mL(-1). After monitoring the immunoreaction signals, the immunocomplex can be easily removed from the APBA interface with a regeneration solution. This regenerated APBA interface can rebound with HRP-anti-CEA and be recognized by the antigen, through which a reusable immunosensor with an RSD of 7.1% for four cycles can be obtained. Under optimal conditions, the detection limit for the CEA immunoassay is 1.1 ng mL(-1), at three times background noise. Serum CEA determination results, obtained with the proposed method, shows that the immunosensor has an acceptable accuracy.