Alpha-fetoprotein In Human Serum Research Articles

Vertically-Ordered Mesoporous Silica Film Based Electrochemical Aptasensor for Highly Sensitive Detection of Alpha-Fetoprotein in Human Serum.

Convenient and rapid detection of alpha fetoprotein (AFP) is vital for early diagnosis of hepatocellular carcinoma. In this work, low-cost (0.22 USD for single sensor) and stable (during 6 days) electrochemical aptasensor was developed for highly sensitive and direct detection of AFP in human serum with the assist of vertically-ordered mesoporous silica films (VMSF). VMSF has silanol groups on the surface and regularly ordered nanopores, which could provide binding sites for further functionalization of recognition aptamer and also confer the sensor with excellent anti-biofouling capacity. The sensing mechanism relies on the target AFP-controlled diffusion of Fe(CN)63-/4- redox electrochemical probe through the nanochannels of VMSF. The resulting reduced electrochemical responses are related to the AFP concentration, allowing the linear determination of AFP with a wide dynamic linear range and a low limit of detection. Accuracy and potential of the developed aptasensor were also demonstrated in human serum by standard addition method.

Unique Electron-Transfer-Mediated Electrochemiluminescence of AuPt Bimetallic Nanoclusters and the Application in Cancer Immunoassay.

Noble Metal nanoclusters (NCs) are promising electrochemiluminescence (ECL) emitters due to their amazing optical properties and excellent biocompatibility. They have been widely used in the detection of ions, pollutant molecules, biomolecules, etc. Herein, we found that glutathione-capped AuPt bimetallic NCs (GSH-AuPt NCs) emitted strong anodic ECL signals with triethylamine as co-reactants which had no fluorescence (FL) response. Due to the synergistic effect of bimetallic structures, the ECL signals of AuPt NCs were 6.8 and 94 times higher than those of monometallic Au and Pt NCs, respectively. The electric and optical properties of GSH-AuPt NCs differed from those of Au and Pt NCs completely. An electron-transfer mediated ECL mechanism was proposed. The excited electrons may be neutralized by Pt(II) in GSH-Pt and GSH-AuPt NCs, resulting in the vanished FL. Furthermore, abundant TEA radicals formed on the anode contributed electrons to the highest unoccupied molecular orbital of GSH-Au2.5Pt NCs and Pt(II), booming intense ECL signals. Because of the ligand effect and ensemble effect, bimetallic AuPt NCs exhibited much stronger ECL than GSH-Au NCs. A sandwich-type immunoassay for alpha fetoprotein (AFP) cancer biomarkers was fabricated with GSH-AuPt NCs as signal tags, which displayed a wide linear range from 0.01 to 1000 ng·mL-1 and a limit of detection (LOD) down to 1.0 pg·mL-1 at 3S/N. Compared to previous ECL AFP immunoassays, this method not only had a wider linear range but also a lower LOD. The recoveries of AFP in human serum were around 108%, providing a wonderful strategy for fast, sensitive, and accurate cancer diagnosis.

Design and preparation of a near-infrared-emissive nanosensor for the quantification and imaging of alpha-fetoprotein in vitro and in vivo

Alpha fetoprotein (AFP) is an embryonic glycoprotein that is produced by hepatocytes and soft yellow sacs, which is closely related to liver cell proliferation, as well as the development of cirrhosis, chronic hepatitis and other diseases. Thus, AFP has been widely used as an important tumor marker in the basic research and clinical diagnosis of diseases such as early-stage liver cancer, endodermal sinus tumors and germ cell tumors. In this study, we developed a near-infrared (NIR) fluorescent carbon quantum dots by microwave synthesis, and constructed a turn-on fluorescent nanosensor by unique modifications. This nanosensor not only has specific response to AFP, but also good biocompatibility and high photostability. The results show that the proposed NIR nanosensor has very high sensitivity, and its detection limit is 3.0 pg/mL for AFP. Thus, it was used for the accurate quantification of AFP in human serum and the imaging of AFP in vitro and in vivo. Therefore, the diagnosis of early-stage liver cancer can be achieved by using proposed method.

An ultrasensitive immunosensor based on cellulose nanofibrils/polydopamine/Cu-Ag nanocomposite for the detection of AFP

In this work, an ultrasensitive immunosensor for amperometric determination of alpha-fetoprotein (AFP) was developed utilizing Ag and Cu nanoparticles on polydopamine (PDA) functionalized cellulose nanofibrils (CNFs) composite (CNFs/PDA/Cu-Ag) as signal amplifier. PDA was first prepared by self-polymerizing of dopamine, and then was adsorbed on CNFs. The obtained CNFs/PDA was applied as substrate to electrolessly deposit Cu-Ag nanoparticles, using NaBH4 as reducing agent. The structure and morphology of the synthesized CNFs/PDA/Cu-Ag nanocomposite were analyzed through Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, X-ray powder diffraction, scanning electron microscopy, particle size analyzer and transmission electron microscopy. The CNFs/PDA/Cu-Ag modified glassy carbon electrode can fix AFP antibody (Ab), and further capture AFP specifically. Electrochemical impedance spectroscopy and cyclic voltammetry were used to characterize the assembly process of immunosensor. The immunoreaction was amplified by electrocatalytical reduction of H2O2 on Cu-Ag nanoparticles, through which AFP was quantitatively detected. The developed sensor exhibits wide linear range of 0.01–100 ng mL−1 (R2 = 0.9963) with low detection limit of 4.27 pg mL−1 (S/N = 3). In addition, it has been used for the detection of AFP in human serum, manifesting its preeminent application prospect in early liver cancer diagnosis.

Reusable, Noninvasive, and Sensitive Fluorescence Enhanced ZnO-Nanorod-Based Microarrays for Quantitative Detection of AFP in Human Serum.

The fabrication of sensitive protein microarrays such as PCR used in DNA microarray is challenging due to lack of signal amplification. The development of microarrays is utilized to improve the sensitivity and limitations of detection towards primal cancer detection. The sensitivity is enhanced by the use of ZnO-nanorods and is investigated as a substrate which enhance the florescent signal to diagnose the hepatocellular carcinoma (HCC) at early stages. The substrate for deposition of ZnO-nanorods is prepared by the conventional chemical bath deposition method. The resultant highly dense ZnO-nanorods enhance the fluorescent signal 7.2 times as compared to the substrate without ZnO-nanorods. The microarray showed sensitivity of 1504.7 ng ml−1 and limit of detection of 0.1 pg ml−1 in wide dynamic range of 0.05 pg-10 μg ml−1 for alpha fetoprotein (AFP) detection in 10% human serum. This immunoassay was successfully applied for human serum samples to detect tumor marker with good recoveries. The ZnO-nanorod substrate is a simple protein microarray which showed a great promise for developing a low-cost, sensitive, and high-throughput protein assay platform for several applications in both fundamental research and clinical diagnosis.

Abstract 1174: Avenue to personalized targeted therapy for hepatoblastoma

Abstract Introduction: Hepatoblastoma (HB) is the most frequent liver malignancy of childhood with an annual incidence of 1.5 per million and a survival rate less than 50% for high-risk, relapse, and treatment refractory patients. The overall goal of this study is to assess the therapeutic efficacies and anti-cancer mechanisms of novel drugs with HB through a drug sensitivity testing pipeline utilizing multiple novel, clinically relevant preclinical patient-derived cell line (PDCL) and patient-derived xenograft (PDX) models. Methods: A patient derived xenograft (PDX) mouse model was generated with intrahepatic implantation of tumor thrombus from a non-metastatic stage 3 patient who recurred after transplantation. A stable PDCL was grown from the murine tumor, and 60 targeted agents were tested for efficacy with the PDCL by using CellTiter-Glo Luminescent Viability assays. Anti-oncogenic effects of the most efficacious agent, dinaciclib (cyclin-dependent kinase (CDK) inhibitor), were further assayed in vitro with HB cell lines (HepG2, HepT1, Huh6, HB17) with phenotypic assays for cell toxicity (MTT assay), proliferation (CCK-8 assay), and anchorage independent colony formation (soft agar assay). Immunoblotting assays were used to assess changes in drug targets and induction of apoptosis (PARP cleavage) with cell lines. Dinaciclib was also tested in vivo in our orthotopic PDX models of high-risk HB. The drug was administered three times a week by intraperitoneal injection (20 mg/kg, 100 µL) for 3 weeks. Magnetic resonance imaging (MRI) and Alpha-fetoprotein (AFP) ELISA were done to evaluate tumor growth throughout the study as tumor-bearing animals show expression of human serum AFP. Tumor weights and volumes, as well as immunohistochemistry for H&E, Ki-67, and TUNEL, were also used to assess effects of drug in vivo. Results: The agent that showed the lowest IC50 value out of 60 tested agents was the CDK inhibitor, dinaciclib (0.7 µM). Dinaciclib suppressed cell proliferation and viability in a dose-dependent manner in all HB cell lines tested with IC50 values in the low micromolar range (HepG2 3.75 µM; HepT1 0.007 µM; Huh-6 1.42 µM; HB17 0.72 µM). Immunoblotting with dinaciclib-treated cells showed induction of PARP cleavage, indicating apoptosis, along with decreased protein expression of CDK1, 2, 5, and 9. Dinaciclib also showed significant in vivo inhibition of tumor growth compared to placebo (relative tumor volume p = 0.03, tumor weight p = 0.02). Conclusions: Drug screening of a novel HB PDCL identified an agent, dinaciclib, that shows preclinical efficacy with refractory disease. Further in vitro and in vivo validation confirmed that dinaciclib is a promising therapeutic agent for the treatment of HB. Such a personalized medicine pipeline of development of PDCLs and testing them with targeted agents will lead to the identification of new drugs that can be used with patients that do not respond to standard therapies. Citation Format: Rohit Kumar Srivastava, Roma Patel, Richard S. Whitlock, Samuel R. Larson, Andrew Badachhape, Sarah E. Woodfield, Sanjeev A. Vasudevan. Avenue to personalized targeted therapy for hepatoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1174.

Generation of a nanobody-alkaline phosphatase fusion and its application in an enzyme cascade-amplified immunoassay for colorimetric detection of alpha fetoprotein in human serum

Sensitive detection of liver disease biomarkers can facilitate the diagnosis of primary hepatoma and other benign liver diseases, and the alpha fetoprotein (AFP) was selected as the model macromolecule in this work. Herein an enzyme cascade-amplified immunoassay (ECAIA) based on the nanobody-alkaline phosphatase fusion (Nb-ALP) and MnO2 nanoflakes was developed for detecting AFP. The bifunctional biological macromolecule Nb-ALP serves as the detection antibody and the reporter molecule. The MnO2 nanoflakes mimic the oxidase for catalyzing the 3,3′,5,5′-tetramethylbenzidine (TMB) into the blue oxidized TMB, which has a quantitative signal at the wavelength of 650 nm. Moreover, the Nb-ALP could dephosphorylate the ascorbic acid-2-phosphate (AAP) to form the ascorbic acid (AA) that can disintegrate the nanoflakes to reduce their oxidation capacity with the content decrease of the oxidized TMB. Using the constructed TMB-MnO2 colorimetric sensing system for Nb-ALP and the optimized experimental parameters, the ECAIA has a limit of detection (LOD) of 0.148 ng/mL which is 18.7-fold lower than that of the p-nitrophenylphosphate (pNPP)-based method (LOD = 2.776 ng/mL). The ECAIA showed good selectivity for AFP with observed negligible cross-reactions with several common cancer biomarkers. The recovery rate for AFP spiked in human serum ranged from 94.8% to 113% with the relative standard deviation from 0.3% to 6.5%. For analysis of the actual human serum samples, a good linear correlation was found between the results tested by the ECAIA and the automatic chemiluminescence analyzer. Thus, the ECAIA was demonstrated to be a promising tool for highly sensitive and selective detection of AFP, providing a reference for analysis of other macromolecule biomarkers.

Construction of Boronate-Affinity Magnetic Immunity SERS Sensor and Detection of Alpha-Fetoprotein (AFP) in Human Serum

Abstract According to its characteristics, borate can recognize and adsorb target glycoproteins and specific binding properties of antigens and antibodies. This article introduces a preparation method for a magnetic immune SERS sensor, and it is used for the specific identification and detection of trace AFP in human serum. The test used AFP as the target protein and 4-mercaptophenylboronic acid (4-MPBA) modified gold-coated silver nanoparticles (Ag@AuNPs) as the signal factor. After the AFP antibody was grafted on the boric acid group, the SERS detection probe (Ag@Au@4-MPBA@Anti-AFP) was obtained. On the surface of boric acid-functionalized (-P) magnetic silica particles, AFP antibody was modified, and the particles were used as magnetic immunocapture probes (Fe3O4@SiO2-P@Anti-AFP). Finally, the sandwich structure complex of “detection probe-target protein-capture probe” was successfully constructed. The prepared magnetic immune SERS sensor exhibits excellent selectivity and high sensitivity for AFP detection; based on the signal-to-noise ratio S/N = 3, the detection limit can reach 1.0 ng/mL; by performing a spike test on AFP in human serum, the sensor can be used to detect AFP in actual samples. The recovery rate was between 85.8% and 105.7%, and the RSD distribution was ≤3.52%. The sensor was easy to prepare, required no complex processing of samples and can achieve rapid detection, so that it is expected to become a new strategy for clinical detection of AFP.

Self-passivated CuV2O6 as a universal photoelectrode material for reliable and accurate photoelectrochemical sensing.

Photoelectrochemical (PEC) detection has attracted intensive attention during the past decade. Currently, most research focuses on improving the sensitivity and selectivity of the PEC sensor, but the issue of the stability of the photoelectrode material under the testing environment is often ignored or lacks in-depth investigation. Herein, we develop a novel CuV2O6 photoelectrode exhibiting superior stability under the testing environment through self-passivation. CuV2O6-based PEC sensors are fabricated for the first time for highly selective carcinoembryonic antigen (CEA) and human serum alpha fetoprotein (AFP) detection. The CuV2O6 shows great potential as a universal photoelectrode material for reliable and accurate PEC detection of macromolecules.

Absolute Quantification of N-Glycosylation of Alpha-Fetoprotein Using Parallel Reaction Monitoring with Stable Isotope-Labeled N-Glycopeptide as an Internal Standard.

Alpha-fetoprotein (AFP) is a well-established serum biomarker for hepatocellular carcinoma (HCC) in clinical laboratories. However, AFP levels can often be high in benign liver diseases such as liver cirrhosis. For this reason, specifically, the level of the aberrant N-glycosylation of AFP has been proposed as a HCC biomarker to improve diagnostic performance using targeted mass spectrometry (MS). In this study, we developed an endoglycosidase-assisted absolute quantification (AQUA) method by which to measure N-glycosylated AFP levels in serum using liquid chromatography-parallel reaction monitoring with immunoprecipitation. Especially, an isotopically labeled synthetic N-glycopeptide with N-acetylhexosamine (HexNAc) attached to asparagine (N) was used as an internal standard. The efficacy of this method was demonstrated by quantifying the N-glycosylation of AFP in human serum. As a result, we showed that the lower limit of the quantification of a stable isotope-labeled N-glycopeptide reached an attomolar level. Our method also had a linear dynamic range from 2 to 6000 ng/mL for N-glycosylated AFP levels. Finally, the N-glycosylation levels of AFP were measured in HCC patients and in healthy donors with the coefficient of variation in both cases (<10% CV). To the best of our knowledge, this is the first report of the AQUA of N-glycosylated AFP in human sera using a stable isotope-labeled glycopeptide as an internal standard. The results demonstrate that our method can facilitate the discovery and verification of aberrant glycoprotein biomarkers in human serum and plasma through sensitive and precise quantification.

Boron nitride quantum dots as electrochemiluminescence coreactants of rGO@Au@Ru–SiO2 for label-free detection of AFP in human serum

A label-free electrochemiluminescence (ECL) immunosensor is developed to detect alpha-fetoprotein (AFP). Ru (bpy)32+-doped silica nanoparticles (Ru–SiO2) and gold nanoparticle-modified reduced graphene oxide (rGO@Au) are integrated to form an rGO@Au@Ru–SiO2 composite. The composite is employed as a carrier for immobilizing the target antibody, and a label-free ECL immunosensor is constructed via antigen–antibody interaction. Owing to the numerous Ru (bpy)32+ molecules in the Ru–SiO2, a high ECL signal is acquired. With excellent conductivity and a large surface area, the rGO@Au composite not only accelerates electron transfer but also improved the ECL signal and captures antibodies, thus enhancing ECL response and sensitivity. Moreover, boron nitride quantum dots (BNQDs) are used as rGO@Au@Ru–SiO2 coreactants, achieving high ECL intensity. The proposed ECL system exhibits significant potential in bioanalysis, which is attributed to the excellent biocompatibility and good stability of BNQDs. Under optimal experimental conditions, the sensor has a linear range of 0.0001 ng mL−1 to 100 ng mL−1 and a limit of detection equal to 0.03 pg mL−1 (S/N = 3). The proposed technique is successfully applied in the susceptible detection of AFP in human serum. The recovery ranges from 99% to 100%, and the RSD is less than 2.8%. The constructed ECL immunosensor exhibits acceptable performance during actual application.

A fluorescent molecularly imprinted device for the on-line analysis of AFP in human serum

MIT is a promising strategy in antibody free analysis for tumour markers. Conventional nanosized MIPs with off-line analysis are beset by tedious operation and unsatisfactory analysis performance. In this work, an on-line analytical device to directly detect AFP, which is a typical tumour marker in cancer screening, was prepared for the first time. A microscope slide was chosen to be the basis of the device. APBA-PA, a polymerizable fluorescent boronic acid monomer, was synthesised and grafted on the surface of the microscope slide to act as the signal transduction pathway between the templates and the device. Along with the hydrolysis of TEOS and the elution of the templates, a portable, stable, easy to operate and low-cost analysis device for AFP with excellent repeatability was successfully prepared. Owing to the excellent selectivity and highly sensitive fluorescence response ability of the device towards the templates, the on-line detection of AFP in human serum was realized. A series of characterizations were applied to the device, and its analysis performance and possible detection mechanism were carefully studied. Furthermore, the device exhibited appropriate application prospects by comparing its analysis results with those of the commercially available ELISA. In our perception, this work is an important step towards MIPs for clinical applications.

A simple immunosensor for alpha-fetoprotein determination based on gold nanoparticles-dextran-reduced graphene oxide

In this paper, a simple and sensitive immunosensor was fabricated for the detection of alpha-fetoprotein (AFP), which is based on a one-pot synthesized nanocomposite of gold nanoparticles-dextran-reduced graphene oxide (AuNPs-Dex-RGO). The AuNPs-Dex-RGO nanocomposite structure was confirmed by transmission electron microscope (TEM), Raman spectroscopy and Fourier transform infrared spectroscopy (FT-IR). With ferricyanide as the electrochemical probe, this method achieved ultrasensitive detection of AFP antigen. The specific immunoreaction between AFP and AFP antibody resulted in a linear relationship between the redox peak current intensity of [Fe(CN)6]4−/3- and AFP concentration. The detection range is 0.01–20ngmL−1, and the detection limit is 0.05pgmL−1. Moreover, the selectivity, reproducibility and stability of the proposed immunosensor were acceptable, which permitted the feasibility for the determination of AFP in human serum with satisfactory results, indicating the great potential for clinical application.

Peptide modified paper based impedimetric immunoassay with nanocomposite electrodes as a point-of-care testing of Alpha-fetoprotein in human serum

Treatment for cancer depends on the type of cancer, and the stage or its development, and thus the need for point-of-care technology that can allow rapid and precise detection of biomarkers is increasing. Here, we present a simple on chip electrical detection of Alpha-fetoprotein (AFP). We rely on using a novel peptide modified plastic-paper microfluidic chips to perform efficient and specific impedimetric detection of AFP in human serum. The chips are prepared from a lower sheet of plastic and upper layer of cellulose chromatography paper modified with silver-20 wt% graphene printed electrodes. Diphenylalanine (FF) was proposed to involve in detection zone of the fabricated microchips in order to improve the sensing performance and the stability of immobilized antibodies according to amine-aldehyde reaction. The target protein is captured on the surface of microchips using specific monoclonal antibodies and the electrical response of the chip is monitored in the presence and absence of different concentrations of AFP. The influence of several parameters including the material types for screen printing of electrodes, FF concentrations, solvent and pH of FF solution on electrical response and cellulose fibers morphology was explored. The impedance measurements of AFP on the fabricated microchip in the optimized parameters exhibited a detection limit of 1 and 10 ng ml−1 in PBS and plasma, respectively. This platform developed here can be adopted to develop systems for rapid detection of biomarkers using portable electric devices.

Direct Monitoring of Fucosylated Glycopeptides of Alpha-Fetoprotein in Human Serum for Early Hepatocellular Carcinoma by Liquid Chromatography-Tandem Mass Spectrometry with Immunoprecipitation.

Alpha-fetoprotein (AFP) is a widely used serological marker that is associated with hepatocellular carcinoma (HCC). Although the level of AFP is increased in HCC, its sensitivity for diagnosis is poor because AFP levels are also increased in liver diseases. Changes in glycoform, especially fucosylation, have been reported to be associated with the development of HCC. The authors introduce the monitoring of fucosylated glycopeptides by liquid chromatography (LC)-mass spectrometry (MS) combined with immunoprecipitation, where glycan-cleaved fragments with an amino acid sequence are used as transitions. Furthermore, neuraminidase for desialylation is useful to improve the MS detection limit (limit of detection [LOD] <2 ng mL-1 ) in 0.1 μL of serum. The performance of the relative percentage of fucosylated AFP (AFP-fuc%) for differentiating between early HCC and cirrhosis is better than that of serum AFP levels as indicated by a greater area under the receiver operator characteristic curve (area under the curve = 0.962 vs. 0.628) and sensitivity (92.3% vs. 53.9%), respectively. Furthermore, the inter- and intraday repeatability of AFP-fuc% in serum is less than 2.1%. These findings suggest that glycopeptide-based LC-MS/MS is a promising method and that AFP-fuc% is a clinically useful parameter for differentiating between early HCC and liver cirrhosis.

Label-free electrochemical aptasensor for detection of alpha-fetoprotein based on AFP-aptamer and thionin/reduced graphene oxide/gold nanoparticles

Sensitive and accurate detection of tumor markers is critical to early diagnosis, point-of-care and portable medical supervision. Alpha fetoprotein (AFP) is an important clinical tumor marker for hepatocellular carcinoma (HCC), and the concentration of AFP in human serum is related to the stage of HCC. In this paper, a label-free electrochemical aptasensor for AFP detection was fabricated using AFP-aptamer as the recognition molecule and thionin/reduced graphene oxide/gold nanoparticles (TH/RGO/Au NPs) as the sensor platform. With high electrocatalytic property and large specific surface area, RGO and Au NPs were employed on the screen-printed carbon electrode to load TH molecules. The TH not only acted as a bridging molecule to effectively capture and immobilize AFP-aptamer, but as the electron transfer mediator to provide the electrochemical signal. The AFP detection was based on the monitoring of the electrochemical current response change of TH by the differential pulse voltammetry. Under optimal conditions, the electrochemical responses were proportional to the AFP concentration in the range of 0.1–100.0 μg/mL. The limit of detection was 0.050 μg/mL at a signal-to-noise ratio of 3. The proposed method may provide a promising application of aptamer with the properties of facile procedure, low cost, high selectivity in clinic.

Preparation of Fe3O4@PS/PDA-Au nanotubes for sensitive electrochemical detection of alpha-fetoprotein

The electrochemical immunosensor was fabricated by immobilizing alpha-fetoprotein (AFP) antigen onto the magnetic glassy carbon electrode (MGCE) that modified by Fe3O4@polystyrene (PS)/polydopamine (PDA)-Au nanotubes. The aim of this study was to develop a new route to prepare Fe3O4@PS/PDA-Au nanotubes with well dispersed Au nanoparticles (AuNPs). The immune detection platform showed good analytical performance for determination AFP in a linear range of 0.01–100ng/mL with a correlation coefficient of 0.9970 under optimal conditions. The detection limit was calculated to 0.0088ng/mL (S/N=3). In addition, the good detection results of AFP in human serum were obtained by the immunosensor. The use of composite nanotubes to build advanced biosensing interface will bring more possibilities for future biomedical detection.

Development of an automated wax-printed paper-based lateral flow device for alpha-fetoprotein enzyme-linked immunosorbent assay

In this study, a novel wax-printed paper-based lateral flow device has been developed as an alternative approach for an automated and one-step enzyme-linked immunosorbent assay (ELISA). The design pattern consisted of a non-delayed channel, a wax-delayed channel, a test zone and a control zone. This system was easily fabricated on a nitrocellulose membrane using a wax-printing method and then baked in an oven at 100°C for 1min. The four barriers of the wax-delayed channel could delay the flow time for 11s compared to the flow time of the non-delayed channel. To use the device under optimal conditions, alpha-fetoprotein (AFP) was detected at a limit of detection of 1ngmL−1 and assessed with the naked eye within 10min. A colorimetric intensity was also measured using a smart phone and computer software at a linear range of 0.1–100ngmL−1 with a good correlation. Furthermore, the proposed device was successfully applied to detect AFP in human serum. Therefore, the wax-printing demonstrates a user-friendly, easy and quick method for the fabrication of the device, which could be used as a one-step, portable, disposable, low-cost, simple, instrument-free and point-of-care device for the automated ELISA.

Gold nanoparticle labeling with tyramide signal amplification for highly sensitive detection of alpha fetoprotein in human serum by ICP-MS

In this work, we developed an immunoassay based on tyramide signal amplification (TSA) and gold nanoparticles (Au NPs) labeling for highly sensitive detection of alpha fetoprotein (AFP) by inductively coupled plasma mass spectrometry (ICP-MS). AFP was captured by anti-AFP1 coating on the 96-well plate and labeled by anti-AFP2-horseradish peroxidase (HRP), in which the HRP can catalyze the deposition of biotinylated tyramine on the nearby protein. Then the streptavidin (SA)-Au NPs was labeled on the deposited biotinylated tyramine as the intensive signal probe for ICP-MS measurement. Under the optimal experimental conditions, the limit of detection of the developed method for AFP was 1.85pg/mL and the linear range was 0.005–2ng/mL. The relative standard deviation for seven replicate detections of 0.01ng/mL AFP was 5.2%. The proposed method was successfully applied to the detection of AFP in human serum with good recoveries. This strategy is highly sensitive and easy to operate, and can be extended to the sensitive detection of other biomolecules in human serum.

An ultra-sensitive label-free immunosensor toward alpha-fetoprotein detection based on three–dimensional ordered IrOx inverse opals

Three-dimensional ordered macroporous iridium oxides (3DOM IrOx 0≤x≤2), were synthesized through a simple self–assembly template method and developed as an electrode material for lable–free immunosensor of cancer biomarker alpha-fetoprotein (AFP). The 3DOM IrOx was well modified on flexible fluorine-doped tin oxide (FTO) electrode and such FTO electrodes based 3DOM IrOx immnuosensor enhanced the adsorption of electrolytes, the diffusion of reactants, and also could act as a rapid electron transfer channel benefited both from the advantage of 3DOM architecture and the nature of IrOx itself. These combined characteristics greatly increased the sensitivity, shortened the response time, reduced the interference, promoted the stability and moreover, demonstrated good accuracy to detect AFP in human serum. The optimum structure and sensing properties of immnuosensor were obtained based on 3DOM IrOx annealed at 500°C. It allows detecting AFP over a wide concentration range from 1 to 250ng/mL with a detection limit of 0.3ng/mL, and a higher slope of the calibration equation. The manufactured immunosensor was also used to determine AFP in human serum with satisfactory results. This kind of 3DOM IrOx modified and FTO-based immunosensor has great application potential for the AFP electrochemical detection.