Enzyme-free Electrochemical Immunosensor Research Articles

An enzyme-free electrochemical immunosensor based on MOF/SWCN nanocomposites for rapid and sensitive detection of fibroblast growth factor 21

An enzyme-free electrochemical immunosensor based on MOF/SWCN nanocomposites for rapid and sensitive detection of fibroblast growth factor 21

Enzyme-free sandwich-type electrochemical immunosensor based on high catalytic binary PdCu mesoporous metal nanoparticles and conductive black phosphorous nanosheets for ultrasensitive detection of pro-SFTPB in non-small cell lung cancer

Pro-surfactant protein B (pro-SFTPB) has been proven to be a powerful biomarker for the early diagnosis of non-small-cell lung cancer (NSCLC). In this study, a novel enzyme-free electrochemical immunosensor is developed for the ultrasensitive detection of pro-SFTPB using binary PdCu mesoporous metal nanoparticles (MMNs) and few-layer black phosphorous (BP nanosheets). Concretely, PdCu MMNs, as novel essential signal labels, were successfully synthesized, offering excellent outstanding peroxidase-like catalytic activity toward hydrogen peroxide (H2O2). Meanwhile, the combination of BP nanosheets with biocompatible gold nanoparticles (AuNPs) served as the substrate on the electrode. With the aid of binary PdCu MMNs nanozymes and BP nanosheets, the enzyme-free electrochemical immunosensor revealed ultrasensitivity detection of pro-SFTPB ranging from 10 pg/mL to 100 ng/mL with an ultralow limit of detection of 5.3 pg/mL. In addition, the immunosensor enables accurate detection of pro-SFTPB in serum samples. Altogether, the developed electrochemical immunosensor provides an enzyme-free platform for pro-SFTPB detection, providing a new method for the early diagnosis of NSCLC.

Determination of E. coli in water using the enzyme free electrochemical impedimetric immunosensors

In this article, we have shown the results of E. coli bacteria determination in water using the enzyme free electrochemical immunosensor with covalent via click chemistry immobilized receptor layer in comparison with dropwise immobilized antibodies. Covalent immobilization was realized by creating precursors on the surface of the working electrode with the reaction of azide-alkyne copper-catalysed cycloaddition approach. The detection limit of the immunosensor with covalent and dropwise immobilization of antibodies was estimated as 6.6 CFU/ml and 11.2 CFU/ml, respectively, a linear range was 103-106 CFU/ml. Moreover, the sensor with “click” immobilized antibodies showed good stability for 30 days when stored in a phosphate buffer, while the sensor with dropwise immobilized receptor layer was stable for 3 days.

An immunosensor based on an electrochemical-chemical-chemical advanced redox cycle amplification strategy for the ultrasensitive determination of CEA

Signal amplification is very important for electrochemical immunoassays. We report an enzyme-free electrochemical immunosensor based on an electrochemical-chemical-chemical (ECC) redox cycle advanced (RCA) signal amplification strategy for the ultrasensitive detection of Carcinoembryonic antigen (CEA). In this scheme, CeO2/Au NPs@SiO2 is connected with ferrocene as the redox indicator, and the detection buffer is composed of the reducing agent hydroquinone (HQ) and tris(2-carboxyethyl) phosphine (TCEP). First, ferrocenecarboxylic acid (FcA) is oxidized to FcA+ on the electrode. Then, HQ reduces FcA+ to FcA to trigger the cyclic reaction of the inner ring. Second, the oxidation product of HQ is catalyzed by TCEP. The product is reduced to HQ again to complete the cyclic reaction of the outer ring, so the entire cyclic reaction forms a closed loop. The system realizes the high-efficiency regeneration of the electroactive material Fc, thereby ensuring the full amplification of the electrical signal. The results show that the immunosensor exhibits good analytical performance, the detection range is 0.01 pg/mL-80 ng/mL, the detection limit is 0.0037 pg/mL, and the immunosensor has excellent selectivity and stability and performs well in the detection of actual samples. This strategy provides a new method for the early screening of CEA.

An enzyme-free electrochemical immunosensor based on quaternary metallic/nonmetallic PdPtBP alloy mesoporous nanoparticles/MXene and conductive CuCl2 nanowires for ultrasensitive assay of kidney injury molecule-1

Herein, a novel enzyme-free electrochemical immunosensor based on quaternary metallic/nonmetallic PdPtBP alloy mesoporous nanoparticles (MNPs)/MXenes and CuCl2 nanowires (NWs) is reported for detection of kidney injury molecule-1 (KIM-1). PdPtBP MNPs/MXene nanocomposites, the novel signal amplification labels, were successfully synthesized using MXenes with the large surface area and the quaternary PdPtBP MNPs nanozymes with outstanding peroxidase-like catalytic activity. Meanwhile, the combination of high conductive CuCl2 NWs with superior biocompatible gold nanoparticles (AuNPs) is introduced as the substrate to modify the glassy carbon electrode (GCE). Benefiting from the excellent electrochemical performances of PdPtBP MNPs/MXene and CuCl2 NWs, the sandwich-type electrochemical immunosensor showed outstanding analytical performance in the presence of hydrogen peroxide (H2O2). Under the optimized conditions, the electrochemical immunosensor exhibited a good linear response from 0.5 ng/mL to 100 ng/mL with a detection limit of 86 pg/mL. Additionally, the established immunosensing method was successfully applied for the specific detection of KIM-1 in the urine. Thus, the enzyme-free electrochemical immunosensor provides a promising prospect for the detection of KIM-1 in the field of clinical diagnostics.

First use of inorganic copper silicate-transduced enzyme-free electrochemical immunosensor for carcinoembryonic antigen detection

Reducing the background signal and interference is an effective method for improving the sensitivity of an electrochemical immunosensor, which is greatly important for detecting tumour markers. In this study, a novel hybrid, nanoflower-like immunosensor probe was synthesised using graded copper silicate hollow spheres (CSH). A ‘close and open’ strategy was used in the immunosensor to magnify response signal. When the reaction conditions were 0.1 M PBS (pH = 7), the silicate and molybdate reaction produced a smaller signal, at which point the signal was off. When the reaction conditions were changed to 0.1 M PBS solution (containing 0.2 M HCl), MoS2 dissolved to form molybdate, while CSH-generated silicates reacted to form molybdate silicates to produce strong oxidation currents and the signal was turned on. This was the first enzyme-free electrochemical reading experiment conducted using inorganic silicate ion transduction. It had the following advantages: (1) CSH spheres with a three-dimensional hierarchical structure had a high specific surface area that allowed them to connect with more antibodies, which improved sensitivity; (2) the method was simple, the detection was fast and the background signal and interference were reduced. The immunosensor had a wide linear range from 0.1 pg mL−1 to 80 ng mL−1 and a minimum detection limit of 0.03 pg·mL−1 (S/N = 3) when detecting carcinoembryonic antigen.

An enzyme-free ultrasensitive electrochemical immunosensor for calprotectin detection based on PtNi nanoparticles functionalized 2D Cu-metal organic framework nanosheets

In this work, a novel high electro-catalytic material was synthesized using PtNi nanospheres to functionalize two-dimensional (2D) ultrathin Cu-TCPP(Fe) nanosheets (PtNi@Cu-TCPP(Fe)), and was further applied to develop an enzyme-free electrochemical immunosensor for the ultrasensitive determination of calprotectin (CALP). The bimetallic Cu-TCPP(Fe) nanosheets with large surface area and massive accessible active sites permitted multiple PtNi to attach their surface, which not only enhanced the catalytic ability and conductivity for non-enzymatic amplification but also provided the modified active sites for antibody immobilization as signal labels. Upon the dual electro-catalytic activity of Cu-TCPP(Fe) and PtNi towards H2O2 reduction for signal amplification, this proposed sandwich CALP immunosensor exhibited a wide linear range of 200 fg mL−1 ∼ 50 ng mL−1 and a low detection limit of 137.7 fg mL−1. The functionalized MOF nanosheets-based biosensing method offered an enzyme-free signal amplification strategy, which provided great promise for further bioanalysis and clinical study.

Enzyme-Free Multiplex Detection ofPseudomonas aeruginosaandAeromonas hydrophilawith Ferrocene and Thionine-Labeled Antibodies Using ZIF-8/Au NPs as a Platform

This is the first work that describes the development of an enzyme-free electrochemical immunosensor for the multiplex detection of Aeromonas hydrophila (Ah) and Pseudomonas aeruginosa (Ps) by usin...

SWCNTs@GQDs composites as nanocarriers for enzyme-free dual-signal amplification electrochemical immunoassay of cancer biomarker

Carcinoembryonic antigen (CEA) as a typical biomarker plays a remarkable role in various human physiological processes. A sensitive and rapid method should be developed to realize the precise diagnosis of cancer at its early stage. In this work, we successfully synthesized the peroxidase-like graphene quantum dots (GQDs) and utilized it to fabricate an enzyme-free electrochemical immunosensor towards CEA by forming a single-wall carbon nanotubes@GQDs (SWCNTs@GQDs) composite. In addition to the SWCNTs@GQDs, reduced graphene oxide-Au nanoparticles (rGO-Au NPs) with the huge specific surface area and excellent conductivity were employed to modify the electrodes, leading to a dual-signal amplification result. Both the electrochemical impedance spectroscopy and cyclic voltammetry measurements were performed to evaluate the interface properties of the layer-by-layer modified electrodes. The proposed electrochemical immunosensor displayed a good specificity and sensitivity towards the detection of CEA ranging from 50 pg mL−1 to 650 pg mL−1 with a low detection limit of 5.3 pg mL−1. This simple and inexpensive immunosensor shows promising potential for the determination of other biomarkers in clinical translation.

Ultrasensitive enzyme-free electrochemical immunosensor for microcystin-LR using molybdenum disulfide/gold nanoclusters nanocomposites as platform and Au@Pt core-shell nanoparticles as signal enhancer

An ultrasensitive enzyme-free electrochemical immunosensor was developed for microcystin- LR (MC-LR) detection based on a unique competitive detection scheme using molybdenum disulfide (MoS2) nanosheets/BSA-stabilized gold nanoclusters (AuNCs) composite and Au core/Pt shell nanoparticles (Au@PtNPs). MoS2/AuNCs nanocomposite was used for a platform for immobilizing more antibody due to its large surface area and excellent biocompatibility. This enzyme-free immunosensor is coupled with a robust immunosandwich format employing Au@PtNPs-labeled antibody as a non-enzymatic reporter. Due to high catalytic efficiency and good stability of Au@PtNPs, Au@PtNPs-immobilized antibody competed for target antigen to form an antibody-antigen immunocomplex and further amplified the electrochemical signal. Thus, the concentration of the MC-LR is directly proportional to the electrochemical signal of immunosensor. Under optimized conditions, the proposed enzyme-free immunosensor was employed to detect MC-LR and exhibited a wide linear range of 1.0 ng L−1–1.0 mg L−1 with low detection limit of 0.3 ng L−1 (S/N = 3). This ultrasensitive and high specific nonenzymatic electrochemical immunoassay shows promising application in environmental and food monitoring.

Electrochemical quantum dots-based magneto-immunoassay for detection of HE4 protein on metal film-modified screen-printed carbon electrodes

A novel enzyme-free electrochemical immunosensor was developed for highly sensitive detection and quantification of human epididymis protein 4 (HE4) in human serum. For the first time, core/shell CdSe/ZnS quantum dots were conjugated with anti-HE4 IgG antibodies for subsequent sandwich-type immunosensing with superparamagnetic microparticles functionalized with anti-HE4 IgG antibodies, which allow rapid and efficient HE4 capture from the sample. Electrochemical detection of anti-HE4 IgG – HE4 – anti-HE4 IgGCdSe/ZnS immunocomplex was performed by recording the current response of Cd(II) ions, released from dissolved quantum dots at screen-printed carbon electrode (SPCE), modified with mercury or bismuth film. The linear range of the detection was from 20 pM to 40 nM with limit of detection of 12 pM using three times the standard deviation of blank criterion at mercury-film SPCE and from 100 pM to 2 nM with limit of detection of 89 pM at bismuth-film SPCE. Proposed electrochemical immunosensor meets the requirements for fast and sensitive quantification of HE4 biomarker in early stage of ovarian cancer and due to the proper sensitivity and specificity presents a promising alternative to enzyme-based probes used routinely in clinical diagnostics.

Multiple amplified enzyme-free electrochemical immunosensor based on G-quadruplex/hemin functionalized mesoporous silica with redox-active intercalators for microcystin-LR detection

A novel multiple amplified enzyme-free immunosensor was developed for competitive immunoassay of microcystin-LR (MC-LR). Classical electrochemical immunosensors usually employ enzymes as biocatalysts to afford amplified signals, but the proteolytic degradation and poor stability are still crucial problem. Herein, monodisperse core-shell mesoporous silica (SiO2@MSN)-functionalized DNAzyme concatamers were synthesized to load hemin and methylene blue (MB) as the mimic enzyme. Firstly, the surface of SiO2@MSN was conjugated with secondary antibody as the recognition of MC-LR antibody and with a DNA strand as the initiator. Two auxiliary DNA strands were then selected for the in-situ propagation to form a double-helix DNA through hybridization chain reaction (HCR), forming numerous DNAzymes (G-quadruplex/hemin) after the addition of hemin. Secondly, MB was inserted into the formed double-helix DNA, and also loaded in the brush-like structure of mesoporous SiO2@MSN. The molecular docking study showed that electrons can transfer more effectively with π-π stack of hemin/G-quadruplex and intercalation of MB/DNA, thus the catalytic ability of DNAzymes can be greatly improved. With the aid of MB, DNAzymes can catalyze the reduction of H2O2 to produce the electrochemical signal. This enzyme-free electrochemical immunosensor can successfully detect MC-LR in a range of 0.5ng/L and 25μg/L with a detection limit of 0.3ng/L. This stable, sensitive and selective nonenzymatic electrochemical immunoassay shows promise for applications in food and environmental monitoring.

Redox active molybdophosphate produced by Cu3(PO4)2nanospheres for enhancing enzyme-free electrochemical immunoassay of C-reactive protein

Redox-active molybdophosphate produced by Cu3(PO4)2nanospheres has been directly employed for signal amplification of an enzyme-free electrochemical immunosensor.

An ultrasensitive enzyme-free electrochemical immunosensor based on redox cycling amplification using methylene blue.

We report a new enzyme-free electrochemical sensor for ultrasensitive measurements of protein biomarkers in plasma and whole blood samples based on a unique electrochemical-chemical-chemical (ECC) redox cycling signal amplification scheme. This scheme uses methylene blue (MB) as a redox indicator which undergoes an endergonic reaction with Ru(NH3)63+ and a highly exergonic reaction with tris(2-carboxyethyl)phosphine (TCEP). This approach offers improved detection sensitivity and sensor stability compared with enzyme-based ECC redox cycling techniques, while involving a simpler sensor modification process and detection protocol. This redox cycling scheme was combined with a robust immunosandwich assay for quantitative measurements of protein biomarkers. For proof of principle, Plasmodium falciparum histidine-rich protein 2 (PfHRP2) was measured in human plasma and whole blood samples, which could be detected down to 10 fg mL-1 and 18 fg mL-1, respectively. Furthermore, this immunosensor exhibits high selectivity, excellent reproducibility and good stability for up to 2 weeks, making it a promising platform for point-of-care testing, especially for detecting extremely low biomarker concentrations in raw biofluids.

Enzyme-free electrochemical immunosensor based on methylene blue and the electro-oxidation of hydrazine on Pt nanoparticles

Enzyme-free electrochemical sensors enable rapid, high sensitivity measurements without the limitations associated with enzyme reporters. However, the performance of non-enzymatic electrochemical sensors tends to suffer from slow electrode kinetics and poor signal stability. We report a new enzyme-free electrochemical immunosensor based on a unique competitive detection scheme using methylene blue (MB), hydrazine and platinum nanoparticles (Pt NPs). This scheme is coupled with a robust immunosandwich format employing a MB-labelled detection antibody as a non-enzymatic reporter. In the presence of the target antigen, surface-immobilized MB consumes interfacial hydrazine thereby diminishing the electro-oxidation of hydrazine on Pt NPs. Thus, the concentration of the antigen is directly proportional to the reduction in the electrochemical signal. For proof-of-concept, this sensor was used to detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), an important malaria biomarker, in unadulterated human saliva samples. Chronocoulometric measurements showed that this platform exhibits pM-range sensitivity, high specificity and good reproducibility, making it well suited for many biosensing applications including noninvasive diagnostic testing.

Ultrasensitive enzyme-free electrochemical immunosensor based on hybridization chain reaction triggered double strand DNA@Au nanoparticle tag

An ultrasensitive enzyme-free electrochemical immunoassay was developed for detection of the fg/mL level carcinoembryonic antigen (CEA) by using a double strand DNA@Au nanoparticle (dsDNA@AuNP) tag and hexaammineruthenium(III) chloride (RuHex) as the electroactive indicator. The dsDNA@AuNP was synthesized by one-pot hybrid polymerization of dsDNA on initiator DNA modified AuNPs via hybridization chain reaction. The immunosensor was prepared by covalently cross-linking capture antibody on chitosan/AuNP nanocomposite modified glass carbon electrode. The AuNPs accelerated the electron transfer and led to high detection sensitivity. With a sandwich-type immunoreaction and a biotin-streptavidin affinity reaction, the dsDNA@AuNP tag was conjugated on the immunocomplex to bring a high amount of RuHex to the electrode surface via electrostatic interaction, resulting in an amplified electrochemical signal. Under optimal conditions, the proposed sensing platform showed a wide linear detection range from 10 fg/mL to 10 ng/mL along with a detection limit of 3.2 fg/mL for CEA. The immunosensor exhibited high sensitivity and good stability, showing a promising application in early cancer diagnosis and could be extended to sensitive electrochemical biosensing of other analytes.

Enzyme-free electrochemical immunosensor configured with Au–Pd nanocrystals and N-doped graphene sheets for sensitive detection of AFP

A novel electrochemical immunosensor capable of enzyme-free detection of alpha fetoprotein (AFP) is reported. This immunosensor was fabricated in a sandwich-like format where catalytic Au–Pd nanocrystals and highly conductive N-doped graphene sheets were incorporated. The significant catalysis by Au–Pd nanocrystals toward hydrogen peroxide, along with the increased electron transfer by graphene sheets, caused signal generation and increased sensitivity, which enables the enzyme-free detection of AFP. With a low detection limit at 0.005ngmL−1, this novel immunosensor worked well over the broad linear range of 0.05–30ngmL−1. Unlike previously reported enzyme-based electrochemical immunosensors, which often involve the complicated steps for enzyme loading and necessary treatments to keep the activity of enzyme, this novel immunosensor is simple in nature and employed catalytic Au–Pd nanoparticles and highly conductive graphene, which thus enables reliable and sensitive detection for clinic usage.

An ultrasensitive enzyme-free electrochemical immunosensor for CA125 using Au@Pd core-shell nanoparticles as labels and platforms for signal amplification.

A facile and sensitive enzyme-free sandwich-type immunosensor for the detection of carbohydrate antigen 125 (CA125) was designed based on Au@Pd core-shell nanoparticles. Au@Pd nanoparticles were used both as electrode-modifying materials to immobilize the primary antibody (Ab1) of CA125 and as labels of secondary antibody (Ab2). The covalent bond between palladium nanoparticles and available amine groups of Ab1 enables the anchoring of larger amounts of Ab1 while the high stability and bioactivity of Ab1 can be reserved. Au@Pd nanoparticles show high electrocatalytic activity toward hydrogen peroxide (H2O2) reduction. The encapsulated Au nanoparticles can also accelerate the electron transfer. A high-performance electrochemical immunosensor was constructed based on the signal amplification of Au@Pd nanoparticles. Under optimal conditions, the immunosensor exhibited a wide linear response to CA125 ranging from 0.002 to 20 U mL-1 with a low detection limit of 0.001 U mL-1. The designed immunosensor displayed an excellent analytical performance with high sensitivity and stability. This strategy was successfully proved to be a simple method, which could be easily extended to other protein analysis.