Photoelectrochemical biosensing platform for the SARS-CoV-2 spike and nucleocapsid proteins

The COVID-19 pandemic is still a continuing worldwide challenge for public health systems. Early and ultrasensitive identification of the infection is essential for preventing the spread of COVID-19 by pre-symptomatic or asymptomatic individuals, particularly in the community and in-home settings. This work presents a versatile photoelectrochemical (PEC) immunosensor for SARS-CoV-2 detection based on a composite material formed by bismuth vanadate (BiVO₄) and strontium titanate (SrTiO₃). The PEC platform was denoted as BiVO₄/SrTiO₃/FTO, and it can be tuned for the detection of either Spike (S) or Nucleocapsid (N) protein by simply altering the antibody immobilized on the platform's surface. Chemical, morphological, and electrochemical characterizations were performed by X-Ray Diffraction, Scanning Electron microscopy, Energy-dispersive X-ray spectroscopy, Electrochemical Impedance Spectroscopy, and Amperometry. With a simple sensing architecture of the PEC platform, it was possible to achieve a linear response range of 0.1 pg mL⁻¹ to 1000 ng mL⁻¹ for S protein and 0.01 pg mL⁻¹ to 1000 ng mL⁻¹ for N protein. The PEC immunosensors presented recovery values for the two SARS-CoV-2 proteins in artificial saliva samples between 97 % and 107.20 % suggesting a good accuracy for the proposed immunosensors.     

Giant Water Uptake Enabled Ultrahigh Proton Conductivity of Graphdiyne Oxide

Proton conductors have attracted great attention in various fields, especially in energy production. Here, we find that graphdiyne oxide (GDYO), derived from graphdiyne (GDY), features the highest proton conductivity of 0.54 S cm⁻¹ (100 % RH, 348 K) among the oxidized carbon allotropes reported so far. The sp- and sp²-co-hybridized carbon skeleton of GDY enables GDYO with the giant water uptake, which is 2.4 times larger than that of graphene oxide (GO), resulting in ultrahigh proton conductivity by increasing the proton concentration and proton conduction pathways. This ultrahigh proton conductivity of GDYO is further proved in a methanol fuel cell by using GDYO membrane as proton exchange membrane. The GDYO membrane enables the cell with higher open circuit voltage, larger power density and lower methanol permeability, compared with commercial Nafion 117. Moreover, the GDYO membrane bears high ion exchange capacity, good acidic stability and low swelling ratio.     

An Ultra-sensitive Electrochemiluminescent Detection of Carcinoembryonic Antigen Using a Hollowed-out Electrode

Ultrasensitive detection of cancer biomarkers has attracted considerable attention recently in academic research and clinic diagnostics. Here, we use a hollowed-out carbon nanotube sponge (CNTSP) electrode to fabricate an immunosensor to realize the sensitive detection of carcinoembryonic antigen (CEA). Nitrogen-doped carbon quantum dots (N-CDs) are combined with antibody that can specially recognize CEA and used as the electrochemiluminescent (ECL) probes in this work. The hollowed-out and permeable CNTSP facilitates chemical species exchange on the surface of electrode, offering an enhanced ECL signal. The resulting ECL immunosensor enables the determination of CEA concentration to be in a wide linear range from 0.005 to 50 pg mL⁻¹ with a detection limit of 1.4 fg mL⁻¹. Furthermore, with good stability, acceptable precision and reproducibility, the proposed ECL assay strategy offers a wide application potential in clinical analysis.     

A Prostate Specific Antigen Immunosensor Based on Biotinylated‐Antibody/Cyclodextrin Inclusion Complex: Fabrication and Electrochemical Studies

Immobilization of biomolecules with a proper orientation is considered as a basis for diverse biotechnological applications. Herein, we report a host‐guest inclusion complexation between β‐cyclodextrin (β‐CD) and biotin as a versatile approach for the immobilization of biomolecules. As a practical application, a sandwich‐type electrochemical immunosensor was designed for the determination of prostate specific antigen (PSA). The immunosensor was fabricated by in situ electropolymerization of poly(N‐acetylaniline) onto a rGO‐modified Pt electrode. Then, β‐CD was covalently grafted onto the over‐oxidized polymer backbone. For improving the efficiency of the assay, AuNPs were casted on the polymeric film, on the surface of which thionine (TH) as an electron mediator was covalently immobilized. Using a host‐guest inclusion complexation between β‐CD and biotin, a β‐CD/biotin‐Ab₁/PSA/Ab₂‐horseradish peroxidase (HRP) sandwich was formed on the electrode surface. The analytical signal was produced via electrochemical reduction of TH_ox, generated by biocatalytic oxidation of the TH_red in the presence of HRP/H₂O₂. Under optimal conditions, the proposed sensor responded linearly to PSA in the range from 10.0 pg mL⁻¹ to 25.0 ng mL⁻¹, with a low detection limit of 6.7 pg mL⁻¹ (S/N=3). Kinetic parameters of the interaction of β‐CD with Ab₁ were also investigated. Finally, the applicability of the immunosensor was successfully investigated for the detection of PSA in human serum samples.     

A Simple and Low-Cost Electrochemical Immunosensor for Ultrasensitive Determination of Calreticulin Biomarker in Human Serum

An early on time detection of breast cancer significantly affects the treatment process and outcome. Herein, a new label-free impedimetric biosensor is developed to determine the lowest change in the level of calreticulin (CALR), which is a new biomarker of breast carcinoma. The proposed immunosensor is fabricated by using reduced graphene oxide/amino substituted polypyrrole polymer (rGO-PPyNH₂) nanocomposite modified disposable electrode. The anti-CALR antibodies are first attached on the rGO-PPyNH₂ nanocomposite coated electrode through glutaraldehyde crosslinking; the CALR antigens are then immobilized with the addition of CALR antigens to form an immunocomplex on the sensing surface. This immunocomplex induces considerably larger interfacial electron transport resistance (R_ct). The variation in the R_ct has a linear relationship with CALR level in the detection range of 0.025 to 75 pg mL⁻¹, with a detection limit of 10.4 fg mL⁻¹. The suggested biosensor shows high selectivity to CALR, good storage stability (at least 5 weeks) and suitable reproducibility results as shown in quality control chart. The designed immunosensor is utilized to analyze CALR levels in human sera with satisfying results. This immunosensor provides a novel way for the clinical determination of CALR and other cancer biological markers.     

Visible Light Photoelectrochemical Sensor for Acetaminophen Determination using a Glassy Carbon Electrode Modified with BiVO4 Nanoparticles

A new and simple photoelectrochemical (PEC) sensor using a glassy carbon electrode (GCE) modified with bismuth vanadate (BiVO₄) nanoparticles and dihexadecyl phosphate (DHP) film was useful for acetaminophen (AC) determination. In 0.2 mol L⁻¹ phosphate buffer (pH=9), the GCE without modification exhibited the smaller photocurrent (0.86 μA) when compared with GCE modified with 1.0 mg mL⁻¹ or 2.0 mg mL⁻¹ BiVO₄ nanoparticles suspension (5.9 and 34 μA, respectively). Based on the photocurrent signal generated through the interaction between GCE, BiVO₄ and the energy of visible light a chronoamperometric method for AC determination was developed. The AC linear range concentration from 0.099 to 0.99 μmol L⁻¹ and limits of detection and quantification of 0.027 and 0.091 μmol L⁻¹, respectively, was obtained. The proposed method was applied to the AC determination in commercial drugs and tap water with satisfactory accuracy and precision. Moreover, the PEC construction was easy and had a short response time, which might confer higher sample throughput for the method.     

Novel visible-light-responsive photoelectrochemical sensor of 2,4-dichlorophenoxyacetic acid using molecularly imprinted polymer/BiOI nanoflake arrays

A novel and highly sensitive visible-light photoelectrochemical (PEC) sensor for the detection of 2,4-D has been developed using a nanocomposite of molecularly imprinted gold nanoparticles-polypyrrole polymer (MIP) modified BiOI nanoflake arrays (BiOINFs) as a photoactive electrode (labeled as MIP@BiOINFs). Our results demonstrate that the smart combination of BiOINFs with MIP offers a high-performance photoactive sensing platform. It features the intrinsically excellent visible-light responsive properties of BiOI and prominent recognition ability from MIP. The designed MIP@BiOINF composite dramatically facilitates the PEC determination of 2,4-D. The detection limit for 2,4-D is found to be as low as about 0.04 ng mL^− 1 (S/N = 3). Moreover, the resulting sensor could be used to detect 2,4-D in spiked soil samples.     

Graphene-based immunosensor for electrochemical quantification of phosphorylated p53 (S15)

We reported a graphene-based immunosensor for electrochemical quantification of phosphorylated p53 on serine 15 (phospho-p53¹⁵), a potential biomarker of gamma-radiation exposure. The principle is based on sandwich immunoassay and the resulting immunocomplex is formed among phospho-p53 capture antibody, phospho-p53¹⁵ antigen, biotinylated phospho-p53¹⁵ detection antibody and horseradish peroxidase (HRP)-labeled streptavidin. The introduced HRP results in an electrocatalytic response to reduction of hydrogen peroxide in the presence of thionine. Graphene served as sensor platform not only promotes electron transfer, but also increases the surface area to introduce a large amount of capture antibody, thus increasing the detection sensitivity. The experimental conditions including blocking agent, immunoreaction time and substrate concentration have been optimized. Under the optimum conditions, the increase of response current is proportional to the phospho-p53¹⁵ concentration in the range of 0.2–10 ng mL⁻¹, with the detection limit of 0.1 ng mL⁻¹. The developed immunosensor exhibits acceptable stability and reproducibility and the assay results for phospho-p53¹⁵ are in good correlation with the known values. This easily fabricated immunosensor provides a new promising tool for analysis of phospho-p53¹⁵ and other phosphorylated proteins.     

Electrochemical immunosensor nanoarchitectonics with the Ag-rGO nanocomposites for the detection of receptor-binding domain of SARS-CoV-2 spike protein

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a grave threat to human life and health, it is essential to develop an efficient and sensitive detection method to identify infected individuals. This study described an electrode platform immunosensor to detect SARS-CoV-2-specific spike receptor-binding domain (RBD) protein based on a bare gold electrode modified with Ag-rGO nanocomposites and the biotin-streptavidin interaction system. The Ag-rGO nanocomposites was obtained by chemical synthesis and characterized by electrochemistry and scanning electron microscope (SEM). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to record the electrochemical signals in the electrode modification. The differential pulse voltammetry (DPV) results showed that the limit of detection (LOD) of the immunosensor was 7.2 fg mL⁻¹ and the linear dynamic detection range was 0.015 ~ 158.5 pg mL⁻¹. Furthermore, this sensitive immunosensor accurately detected RBD in artificial saliva with favorable stability, specificity, and reproducibility, indicating that it has the potential to be used as a practical method for the detection of SARS-CoV-2.

     

Resonance energy transfer between ZnCdHgSe quantum dots and gold nanorods enhancing photoelectrochemical immunosensing of prostate specific antigen

Gold nanorods (AuNRs) integrated with ZnCdHgSe near-infrared quantum dots (AuNRs-ZnCdHgSe QDs) were successfully synthesized and characterized by transmission electron microscope, X-ray photoelectron spectroscopy, and X-ray diffraction. A glassy carbon electrode was decorated with the aforementioned AuNRs-ZnCdHgSe QDs nanocomposite, which provides a biocompatible interface for the subsequent immobilization of prostate specific antibody (anti-PSA). After being successively treated with glutaraldehyde vapor and bovine serum albumin solution, a photoelectrochemical immunosensing platform based on anti-PSA/AuNRs-ZnCdHgSe QDs/GCE was established. The photocurrent response of ZnCdHgSe QDs was tremendously improved by AuNRs due to the effect of resonance energy transfer which can be deduced from the dependence of the enhanced efficiency on the AuNRs with different length-to-diameter ratios and spectral absorption characteristics. A maximum photocurrent was obtained when the absorption spectrum of AuNRs matched well with the emission spectrum of ZnCdHgSe QDs. A photoelectrochemical immunosensor for prostate specific antigen (PSA) was achieved by monitoring the photocurrent variation. The photocurrent variation before and after being interacted with PSA solution exhibits a good linear relationship with the logarithm of its concentration (logc_PSA) in the range from 1.0 pg mL⁻¹ to 50.0 ng mL⁻¹. The detection limit of this photoelectrochemical immunosensor is able to reach 0.1 pg mL⁻¹ (S/N = 3). Determining PSA in clinical human serum was also demonstrated by using the developed anti-PSA(BSA)/AuNRs-ZnCdHgSe QDs/GCE electrode. The results were comparable with those obtained from an enzyme-linked immunosorbent assay method.     

Electrochemical Immunosensor Made with Zein-based Nanofibers for On-site Detection of Aflatoxin B1

A disposable electrochemical immunosensor for on-site detection of aflatoxin B1(AFB1), one of the most toxic mycotoxins in agri-food products, was fabricated through a low-cost cut-printing method and then modified with zein/polypyrrole(PPy) electrospun nanofibers onto which anti-AFB1 monoclonal antibodies were immobilized covalently. Fabrication was possible with an innovative and simple approach to adsorb nanofibers onto the working electrode during electrospinning. Electrochemical impedance spectroscopy was employed as the principle of detection, and the data collected with a portable potentiostat were treated with information visualization techniques. The nanostructured immunosensor showed a high sensitivity for AFB1 with a linear detection range from 0.25 to 10 ng mL⁻¹ and a theoretical limit of detection of 0.092 ng mL⁻¹, which is adequate to detect AFB1 in food, according to regulatory agencies.     

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。     

New Insights on Potentiometric Immunosensor at Carbon Fiber Microelectrode for Alpha-Fetoprotein in Hepatocellular Carcinoma

Herein, we investigated the analytical features of potentiometric immunosensors for detection of alpha-fetoprotein (AFP) in hepatocellular carcinoma at different electrodes, such as carbon fiber microelectrode (CFME) and carbon-disk electrode (CDE), respectively. To construct such an immunosensor, anti-AFP capture antibodies were first conjugated covalently onto the activated electrodes through typical carbodiimide coupling. Thereafter, one-step immunoreaction protocol was successfully introduced to develop a new potentiometric immunoassay upon addition of AFP. Accompanying the antigen-antibody reaction, the surface charges of the modified electrodes were changed for the readout of electric potential. Results indicated that the linear range of CDE-based immunosensor was 0.1–100 ng mL⁻¹ AFP, whereas the assay sensitivity by using CFME could be further increased to 3.2 pg mL⁻¹ with the linear range from 0.01 to 500 ng mL⁻¹ AFP. Meanwhile, CFME-based immunosensor showed high sensitivity, good reproducibility and specificity, and could be utilized for the analysis of human serum specimens with consistent results relative to commercialized ELISA kit.     

A Highly Selective Poly(thiophene)‐graft‐Poly(methacrylamide) Polymer Modified ITO Electrode for Neuron Specific Enolase Detection in Human Serum

In this study, an impedimetric immunosensor based on polymer poly(thiophene)‐graft‐poly(methacrylamide) polymer (P(Thi‐g‐MAm)) modified indium tin oxide (ITO) electrode is developed for the detection of the Neuron Specific Enolase (NSE) cancer biomarker. First, the P(Thi‐g‐MAm) polymer is synthesized and coated on the ITO electrode by using a spin‐coating technique. P(Thi‐g‐MAm) polymer acts as an immobilization platform for immobilization of NSE‐specific monoclonal antibodies. Anti‐NSE antibodies are utilized as biosensing molecules and they bind to the amino groups of P(Thi‐g‐Mam) polymer via glutaraldehyde cross‐linking. Spin‐coating technique is employed for bioelectrode fabrication and this technique provides a thin and uniform film on the ITO electrode surface. This bioelectrode fabrication technique is simple and it generates a suitable platform for large‐scale loadings of anti‐NSE antibodies. This immunosensor exhibits a wide linear detection range from 0.02 to 4 pg mL^?1 and with an ultralow detection limit of 6.1 fg mL^?1. It reveals a good long‐term stability (after 8 weeks, 78% of its initial activity), an excellent reproducibility (1.29% of relative standard deviation (RSD)), a good repeatability (5.55% of RSD), and a high selectivity. In addition, the developed immunosensor is proposed as a robust diagnostic tool for the clinical detection of NSE and other cancer biomarkers.     

Multiplexed Determination of Fertility-related Hormones in Saliva Using Amperometric Immunosensing

This paper reports a dual immunosensor for the simultaneous determination of two important fertility-related hormones: 17β-estradiol, E2, and follicle-stimulating hormone, FSH. The implemented method involves direct competitive (E2) or sandwich-type (FSH) immunoassays carried out on magnetic microparticles (MBs) and amperometric detection at screen-printed dual carbon electrodes (SPdCEs) involving the hydroquinone (HQ)/H₂O₂ system. The developed immune platform demonstrates LOD values of 6.88 pg mL⁻¹ and 0.11 mIU mL⁻¹ for E2 and FSH standards, respectively and usefulness for the determination in saliva samples collected from different volunteers, giving results in agreement with the conventional ELISA methodologies.     

Amplified Electrochemical Immunosensor for Calmodulin Detection Based on Gold‐Silver‐Graphene Hybrid Nanomaterials and Enhanced Gold Nanorods Labels

Calmodulin (CaM) is an important intracellular calcium‐binding protein. It plays a critical role in a variety of biological and biochemical processes. In this paper, a new electrochemical immunosensing protocol for sensitive detection of CaM was developed by using gold‐silver‐graphene (AuAgGP) hybrid nanomaterials as protein immobilization matrices and gold nanorods (GNRs) as enhanced electrochemical labels. Electrode was first modified with thionine‐chitosan film to provide an immobilization support for gold‐silver‐graphene hybrid nanomaterials. The hybrid materials formed an effective matrix for binding of CaM with high density and improved the electrochemical responses as well. Gold nanorods were prepared for the fabrication of enhanced labels (HRP‐Ab₂‐GNRs), which provided a large capacity for HRP‐Ab₂ immobilization and a facile pathway for electron transfer. With two‐step immunoassay format, the HRP‐Ab₂‐GNRs labels were introduced onto the electrode surface, and produced electrochemical responses by catalytic reaction of HRP toward enzyme substrate of hydrogen peroxide (H₂O₂) in the presence of thionine. The proposed immunosensor showed an excellent analytical performance for the detection of CaM ranging from 50 pg mL^?1 to 200 ng mL^?1 with a detection limit of 18 pg mL^?1. The immunosensor has also been successfully applied to the CaM analysis in two cancer cells (HepG2 and MCF‐7) with high sensitivity, which has shown great potency for improving clinic diagnosis and treatment for cancer study.     

A photoelectrochemical immunosensor based on Au-doped TiO2 nanotube arrays for the detection of α-synuclein

α‐Synuclein (α‐SYN) is a very important neuronal protein that is associated with Parkinson’s disease. In this paper, we utilized Au‐doped TiO₂ nanotube arrays to design a photoelectrochemical immunosensor for the detection of α‐SYN. The highly ordered TiO₂ nanotubes were fabricated by using an electrochemical anodization technique on pure Ti foil. After that, a photoelectrochemical deposition method was exploited to modify the resulting nanotubes with Au nanoparticles, which have been demonstrated to facilitate the improvement of photocurrent responses. Moreover, the Au‐doped TiO₂ nanotubes formed effective antibody immobilization arrays and immobilized primary antibodies (Ab₁) with high stability and bioactivity to bind target α‐SYN. The enhanced sensitivity was obtained by using {Ab₂‐Au‐GOx} bioconjugates, which featured secondary antibody (Ab₂) and glucose oxidase (GOx) labels linked to Au nanoparticles for signal amplification. The GOx enzyme immobilized on the prepared immunosensor could catalyze glucose in the detection solution to produce H₂O₂, which acted as a sacrificial electron donor to scavenge the photogenerated holes in the valence band of TiO₂ nanotubes upon irradiation of the other side of the Ti foil and led to a prompt photocurrent. The photocurrents were proportional to the α‐SYN concentrations, and the linear range of the developed immunosensor was from 50 pg mL^?1 to 100 ng mL^?1 with a detection limit of 34 pg mL^?1. The proposed method showed high sensitivity, stability, reproducibility, and could become a promising technique for protein detection.     

Label‐free Electrochemical Immunosensor for Cardiac Troponin T Based on Exfoliated Graphite Nanoplatelets Decorated with Gold Nanoparticles

This paper describes the application of exfoliated graphite nanoplatelets (xGnP) decorated with gold nanoparticles (AuNP) for the development of a label‐free electrochemical immunosensor for the determination of human cardiac troponin T (TnT), an important cardiac biomarker in the diagnosis of acute myocardial infarction (AMI). Heparin‐stabilized AuNP (AuNP‐Hep) were synthesized, characterized and supported on xGnP. The material obtained (AuNP‐Hep‐xGnP) was used as a platform to immobilize the anti‐TnT by adsorption and this was then applied in the construction of an immunosensor. Under optimized conditions, using differential pulse voltammetry (DPV) and an incubation time of 20 min, the proposed immunosensor showed linearity in the range of 0.050 to 0.35 ng mL⁻¹ TnT, with a calculated limit of detection of 0.016 ng mL⁻¹. The interday precision (n=7) showed a coefficient of variation of 6.5 %. Some potential interferents commonly present in blood plasma samples were investigated and the degree of interference was found to be low (less than 10 %), demonstrating adequate selectivity for analytical applications. The biosensor was successfully applied in the determination of TnT in fortified samples of human blood plasma.     

Self-powered anti-interference photoelectrochemical immunosensor based on Au/ZIS/CIS heterojunction photocathode with zwitterionic peptide anchoring

Accurate detection of important biomarkers with ultra-low levels in complex biological matrix is one of the frontier scientific issues because of possible signal interference of potential reductive agents and protein molecules. Herein, a self-powered anti-interference photoelectrochemical (PEC) immunosensor was explored for sensitive and specific detection of model target of cardiac troponin I (cTnI). Specifically, a novel ternary heterojunction served as the photocathode to offer a remarkable current output and a zwitterionic peptide was introduced to build a robust antifouling biointerface. CuInS₂ (CIS) film with porous network nanostructure was first prepared and then modified in order with ZnIn₂S₄ (ZIS) nanocrystals and Au nanoparticles to fabricate the Au/ZIS/CIS heterojunction photocathode. After capture cTnI antibody (Ab) was immobilized, the zwitterionic peptide KAEAKAEAPPPPC was then anchored to compete the immunosensor. The elaborated PEC immunosensor exhibited high sensitivity for target cTnI antigen (Ag) detection, with good anti-interference against reductive agents and nonspecific proteins. This integration strategy of heterojunction photocathode with zwitterionic peptide provides a new sight to develop advanced PEC immunosensors applying in practical biosamples.     

An electrochemical biosensor for α-fetoprotein based on carbon paste electrode constructed of room temperature ionic liquid and gold nanoparticles

A novel and effective electrochemical immunosensor for the rapid determination of α-fetoprotein (AFP) based on carbon paste electrode (CPE) consisting of room temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate (BPPF₆) and graphite. The surface of the CPE was modified with gold nanoparticles for the immobilization of the α-fetoprotein antibody (anti-AFP). By sandwiching the antigen between anti-AFP on the CPE modified with gold nanoparticles and the secondary antibody, polyclonal anti-human-AFP labeled with horseradish peroxidase (HRP-labeled anti-AFP), the immunoassay was established. The concentration of AFP was determined based on differential pulse voltammetry (DPV) signal, which was generated in the reaction between O-aminophenol (OAP) and H₂O₂ catalyzed by HRP labeled on the sandwich immunosensor. AFP concentration could be measured in a linear range of 0.50-80.00 ng mL⁻¹ with a detection limit of 0.25 ng mL⁻¹. The immunosensor exhibited high sensitivity and good stability, and would be valuable for clinical assay of AFP.