Ultrasensitive Detection Of Alpha-fetoprotein Research Articles

A novel microfluidic chip integrating with microcolumn array electrodes for rapid and ultrasensitive detection of alpha-fetoprotein

BackgroundCancer posed a serious threat to human health, and early diagnosis of cancer biomarker was extremely important for the treatment and control of cancer. Electrochemistry-based assays were low-cost, responsive and easy to operate, but there were some challenges in terms of accuracy, detection limit, efficiency and portability. The combination of microfluidic devices and electrochemical methods was expected to construct a high-performance sensing platform, but long-time antigen-antibody incubation was still required. Therefore, a novel microfluidic chip needs to be developed, which has the advantages of good portability, short incubation time, high accuracy, low detection limit and great application to point-of-care testing. ResultsA microfluidic sensor based on microcolumn array electrodes was developed, in which microcolumns could create local mixed flow to reduce the incubation time of target molecules and enhance their interaction with the sensing interface. Besides, three dimensional Mxene fibers-gold nanoparticles (3D MF-Au) was modified on the microcolumn array electrodes to increase active sites and provide more electrolyte shuttle holes. The electrolyte turbulence caused by the microcolumn array electrodes could heighten the contact between the target molecules and sensing interface and accelerate the transfer of redox pairs, thus reducing the incubation time of the target molecules and improving the electrochemical responses in synergy with the 3D MF-Au. Herein, the detection of AFP was chosen as a model, and the microfluidic sensor possessed superior performance for analysis of AFP in the range of 0.1 pg mL−1 - 200 ng mL−1 with a low detection limit (LOD) of 0.0648 pg mL−1. SignificanceThis microfluidic chip integrating with microcolumn array electrodes has been successfully implemented to detect AFP in human serum, and the results were consistent with that of electrochemical chemiluminescence method. The microfluidic chip provided a new strategy of portability, shortening incubation time and enhancing electrical signals for antigen detection of real samples, which showed great utilization potentiality in point-of-care testing.

A novel microfluidics PMMA/paper hybrid bioimmunosensor for laser-induced fluorescence detection in the determination of alpha-fetoprotein from serum

Low loss and high capture efficiency of target samples are prerequisites for sensitive detection by fluorescence immunosensors. For quantitative and ultrasensitive detection of alpha-fetoprotein (AFP), we designed a novel microfluidics pump-free PMMA/paper hybrid immunosensor. Here, the 2D planar structure of the detection area of the plastic microfluidic chip is improved to a 3D spatial structure, which increases the number of capture antibodies immobilized and the cross-section area of the “intercept” target protein. PMMA channels efficiently transport target proteins. The paper-based, three-dimensional detection area not only increases the amount of immobilization of the captured antibody but also improves the contact probability between the captured antibody and the target protein. This greatly improves the efficiency of immune binding. A laser-induced fluorescence detector is applied to detect the target protein on the paper-based detecting unit. Under optimal conditions, the immunosensor ensures that the reaction time is 12 min and the residual liquid is only 6.1% of the original volume, achieving ultra-sensitive detection of AFP in the range of 0.01 ng/mL to 100 ng/mL with a limit of quantitation (LOQ) as low as 4.5 pg/mL. The recoveries for AFP ranged from 96.6% to 101.5%, which means the hybrid microfluidics chip is reliable and has great potential for early screening.

Construction of a competitive electrochemical immunosensor based on sacrifice of Prussian blue and its ultrasensitive detection of alpha-fetoprotein

Effective signal amplification is a prerequisite for ultrasensitive detection by electrochemical immunosensors. For quantitative and ultrasensitive detection of alpha-fetoprotein (AFP), we designed a competitive electrochemical immunosensor and transferred the immunoreactivity from the electrode surface to the cuvette. AFP antigen was captured using AFP primary antibody (Ab1) immobilized on magnetic nanobeads (MBs), and ZIF-8 nanomaterials attached to secondary antibody (Ab2) were used as probes. MBs helped retain the sandwich structure in the test tube through incubation and washing steps. Then, an appropriately fixed excess of sodium ethylenediaminetetraacetic acid (EDTA) solution was added to the cuvettes, resulting in etching of Zn ions from ZIF-8 and formation of Zn-EDTA complexes. After magnetic separation, a certain amount of supernatant is added dropwise to the Prussian blue (PB)-modified electrode (GCE), and Fe ions (from PB) complex with the remaining EDTA in the supernatant, thus reducing the signal response value of PB. The higher the AFP concentration, the lower the amount of free EDTA in the supernatant, the less the destruction of PB, and therefore the higher the current. Under optimal conditions, the immunosensor achieved ultra-sensitive detection of AFP in the range of 10−4 ng/mL-100 ng/mL with a limit of detection (LOD) as low as 0.032 pg/mL (S/N = 3). The excellent performance provides an important tool for the early screening and detection of AFP.

An innovative electrochemical immunosensor based on nanobody heptamer and AuNPs@ZIF-8 nanocomposites as support for the detection of alpha fetoprotein in serum

A novel electrochemical immunoassay based on nanobody heptamer for ultrasensitive detection of alpha fetoprotein (AFP) is proposed. First, AFP heptamer fusion protein (A1-C4bp α) is prepared via fusing the AFP-specific nanobody (A1) with the C-terminal fragment of C4-binding protein (C4bp α). Then A1-C4bp α heptamer with higher affinity is used as the molecule recognizing probe to capture numerous the AFP, increasing the sensitivity of the immunosensor. Moreover, AuNPs decorated zeolitic imidazolate framework (AuNPs@ZIF-8) with large surface area, and abundant binding sites serves an ideal nanocarrier to increase heptamer loading. Multiwalled carbon nanotube (MWCNTs) with excellent electrical conductivity was immobilized on glassy carbon electrode (GCE) to enhance electrochemical signals. The coupling between the AuNPs@ZIF-8@MWCNTs and heptamer technologies constructed an ultrasensitive and specific immunosensor for AFP detection in the range of 0.1 to 105 pg mL−1 with a lower detection limit of 0.033 pg mL−1. The as-prepared electrochemical sensing exhibited excellent performance and could be employed to determine AFP with satisfactory results in actual serum samples, suggesting that the proposed strategy for nanobody multimerization provides an alternative scheme for enhancing sensitivity in important biomarkers detection.

Nonlinear Hybridization Chain Reaction Integrated with Fluorescent Immunosensor for Ultrasensitive Detection of Alpha Fetoprotein

Nonlinear Hybridization Chain Reaction Integrated with Fluorescent Immunosensor for Ultrasensitive Detection of Alpha Fetoprotein

Preparation of an electrochemical immunosensor based on a Cu/Cu2O-rGO@Au signal synergistic amplification strategy and efficient and sensitive detection of alpha-fetoprotein.

The effective amplification of the signal is the prerequisite for the ultrasensitive detection of electrochemical immunosensors. To quantitatively detect alpha-fetoprotein (AFP), we prepared a sandwich-type electrochemical immunosensor. Using a gold nanoparticles (Au NPs) modified glassy carbon electrode (GCE) as the sensing platform and Cu/Cu2O-rGO@Au as the signal label, differential pulse voltammetry (DPV) was used to achieve sensitive detection of AFP. We found that the nanomaterials can undergo electro-oxidation and electro-reduction reactions between Cu(I) and Cu(II) in a buffer solution of pH = 6.0. It is worth mentioning that the incorporation of metals into metal oxide substrates is a new strategy to combine the catalytic activity of metal oxides with the electrical conductivity of metals. Reduced graphene oxide (rGO), which is rich in oxygen-containing groups, can load more Cu/Cu2O and Au NPs and increase the conductivity. The modification of Au NPs makes them have better biocompatibility and conductivity. Under the best detection conditions, the prepared immunosensor realizes the specific and ultrasensitive detection of AFP. The detection range is 0.01-50 ng mL-1 and the limit of detection (LOD) was as low as 0.589 pg mL-1 (S/N = 3); meanwhile it also has good practical application ability. Therefore, this immunosensor provides an important means for the early screening and detection of AFP.

Electrochemical immunosensor using artificial enzyme-induced metallization for the ultra-sensitive detection of alpha fetoprotein

During the long incubation process of an electrochemical immunosensor, the sensitivity and detection limit of the sensor are seriously affected because the signal label easily falls off. In this work, we proposed an innovative artificial enzyme-induced metallization process. Firstly, an artificial enzyme is used to catalyze the solution to produce a reducing substance. Subsequently, metal ions in the solution are reduced to the surface of the electrode. Finally, the metal is dissolved by electrochemically, and the peak current is proportional to the alpha-fetoprotein (AFP) concentration. As a Lewis acid, CeO2 can hydrolyze phosphate bonds, and the hydrolysate ascorbic acid (AA) can reduce silver nanoparticles. In addition, the Au@CeO2 yolk shell nanostructure (Au@CeO2 YSNs) can greatly improve the catalytic activity of CeO2 and the conductivity of the signal tag. More importantly, since the signal substance is deposited on the electrode surface as soon as it is generated, the loss of electrical signals is reduced. Results show that the immunosensor has good analytical performance, a detection range of 0.1 pg/mL to 200 ng/mL, a detection limit of 0.035 pg/mL, and it performs well in serum samples. Therefore, this strategy provides a novel method for the early screening of AFP.

Ultrasensitive SERS-Based Immunoassay of Tumor Marker in Serum Using Au–Ag Alloy Nanoparticles and Ag/AgBr Hybrid Nanostructure

Ultrasensitive detection of alpha-fetoprotein (AFP) is critical for the early diagnosis of liver cancer. In this work, a novel surface-enhanced Raman scattering (SERS)-based immunoassay complex has been successfully developed for the detection of AFP by using the Au-Ag alloy nanoparticals and the Ag/AgBr hybrid nanostructure. As the typical bimetal or metal/semiconductor plasmonic materials, besides the strong SERS enhancement characteristics, the Au-Ag alloy nanoparticals exhibit excellent monodispersity and the Ag/AgBr hybrid nanostructure demonstrates good stability. The experimental results show that the SERS-based immunoassay of AFP presents a low limit of detection of 1.86[Formula: see text]fg/mL and a broad dynamic range from 2[Formula: see text]fg/mL to 0.8[Formula: see text][Formula: see text]g/mL. Furthermore, the clinical applicability of the proposed SERS-based immunoassay has been assessed by the detection of AFP in the human serum samples of cancer patient and healthy person. The test data are consistent well with that of chemiluminescence immunoassay (CLIA) in the relative errors of [Formula: see text]8.82–8.06% and show better detection sensitivity. It reveals that the proposed immunoassay protocol is significant for giving insight into the design of ultrasensitive biosensor and the point-of-care testing of cancers.

Enzyme–antibody dual-film modified gold nanoparticle probe for ultrasensitive detection of alpha fetoprotein

In this study, we designed a comprehensive strategy for the ultrasensitive detection of alpha fetoprotein (AFP) with high specificity using gold nanoparticle (AuNP)-based enzyme-linked immunosorbent assay (ELISA). A dual-film modified probe was synthesized by coating AuNPs with horseradish peroxidase (HRP) on its surface. Anti-AFP monoclonal antibody (McAb) was immobilized on the surface of the enzyme using glutaraldehyde cross-linking method. AuNPs, employed as support for the immobilization of HRP. HRP was used not only as the enzymatic-amplified tracer but also as a bridge for loading McAb. The limit of detection was 2 ng mL-1. The developed probes can provide an alternative approach with high sensitivity and a simple process similar to that of the traditional HRP-McAb based ELISA for the ultrasensitive detection of AFP in serum.

Coagulation induced attenuation of plasmonic absorption of Au nanorods: Application in ultra sensitive detection of alpha-fetoprotein

The effect of α-fetoprotein (AFP) on the plasmonic absorption spectra of colloidal gold nanorods (GNRs) has been investigated experimentally. Because of the AFP induced aggregation and coagulation, both the intensity and wavelength of the longitudinal surface plasmon resonance (SPR) peak are sensitive to the AFP concentration. It has been found the absorption intensity always decreases monotonously as the AFP concentration is increased. By analyzing the intensity fading of the absorption peak, AFP could be detected with a sensitivity of 0.25pg/mL and an ultra low limit of 0.25pg/mL. What's more, the sensitivity and detection limit could be further improved on the condition of pre-centrifugation of the colloidal GNRs. The corresponding mechanism has also been discussed. Cetyl trimethyl ammonium bromide (CTAB) molecules play the important role in preventing the coagulation of GNRs and affect the AFP detection.

Electrochemiluminescence immunosensor based on graphene–CdS quantum dots–agarose composite for the ultrasensitive detection of alpha fetoprotein

A novel strategy for the enhancement of electrochemiluminescence (ECL) was developed by combining CdS quantum dots (QDs), graphene (G) and agarose. This enhanced ECL was exploited to develop a label-free ECL immunosensor for the ultrasensitive detection of alpha fetoprotein (AFP). The novel G–CdS QDs–agarose composite was first coated on the glass carbon electrode surface to form a robust film, which exhibited high ECL intensity, good biocompatibility and high stability. After that 3-aminopropyl-triethoxysilane (APS), as a binding linker, was conjugated to the G–CdS QDs–agarose composite film on the electrode, the ECL signal was significantly enhanced. The fabrication of ECL immunosensor was successfully completed by immobilizing the AFP-antibody (Ab) onto the electrode through glutaric dialdehyde (GLD). The specific immunoreaction between AFP and antibody resulted in the decrease in ECL intensity and the intensity decreased linearly with the logarithm of AFP concentration in the range of 0.0005–50pgmL−1 with a detection limit of 0.2fgmL−1. The immunosensor exhibits high sensitivity, specificity, stability, reproducibility and good regeneration, thus has the potential to be used in clinical application. Besides, the highly enhanced ECL from the G–CdS QDs–agarose composite film opened new avenues to apply graphene and QDs ECL in analytical systems and ECL biosensors.

Electrochemiluminescence immunosensor for the determination of ag alpha fetoprotein based on energy scavenging of quantum dots

An electrochemiluminescence (ECL) immunosensor for ultrasensitive detection of alpha fetoprotein (AFP) was fabricated using graphene-CdS quantum dots-alginate (G-CdS QDs-AL) as the immobilizing support and CdSe/ZnS QDs as the label. CdSe/ZnS QDs could effectively scavenge the ECL of G-CdS QDs-AL composite, and the quenched ECL intensity depended linearly on the logarithm for AFP concentration in the range from 0.05 to 500 fg/mL. The detection limit was 20 ag/mL. The proposed ECL immunoassay protocol for AFP detection is stable, specific, highly sensitive and promising for clinical application.