Ratiometric Immunosensor Research Articles

L-Cysteine-Tuned the Hierarchical Structure Based on Benzimidazole: Synthesis, Characterization, and Application in Ratiometric Electrochemiluminescence Immunoassay.

Efficient and robust electrochemiluminescence (ECL) emitters are crucial for enhancing the ECL immunosensor sensitivity. This study introduces a novel ECL emitter, CoBIM/Cys, featuring a hierarchical core-shell structure. The core of the structure is created through the swift coordination between the sulfhydryl and carboxyl groups of l-cysteine (l-Cys) and cobalt ions (Co2+), while the shell is constructed by sequentially coordinating benzimidazole (BIM) with Co2+. This design yields a greater specific surface area and a more intricate porous structure compared to CoBIM, markedly enhancing mass transfer and luminophore accessibility. Moreover, the l-Cys and Co2+ core introduces Co-S and Co-O catalytic sites, which improve the catalytic decomposition of H2O2, leading to an increased production of hydroperoxyl radicals (OOH•). This mechanism substantially amplifies the ECL performance. Leveraging the competitive interaction between isoluminol and BIM for OOH• during ECL emission, we developed a ratiometric immunosensor for cardiac troponin I (cTnI) detection. This immunosensor demonstrates a remarkably broad detection range (1 pg mL-1 to 10 ng mL-1), a low detection limit (0.4 pg mL-1), and exceptional reproducibility and specificity.

A novel ratiometric electrochemical immunosensor for the detection of cancer antigen 125 based on three-dimensional carbon nanomaterial and MOFs

Accurate and sensitive detection of cancer antigen 125 (CA125) is important in early ovarian cancer diagnosis and monitoring. In the present work, a novel electrochemical ratiometric immunosensor for the determination of CA125 based on three-dimensional reduced graphene oxide/multi-walled carbon nanotube carboxylic acid- thionine (3DrGO/MWCNTs-Thi) and UIO-66-NH2/ferrocenecarboxylic acid (UiO-66-Fc) was constructed. 3DrGO/MWCNTs-Thi of immobilized primary antibody (Ab1) and UiO-66-Fc of labeled second antibody (Ab2) were used as capture probes and signaling probes for CA125, respectively. As the concentration of CA125 increases, the oxidation peak currents (IThi and IFc) generated by the two materials were changed in opposite directions, and therefore the ratio of the currents (IThi/IFc) was negatively correlated with the concentration of CA125. Thanks to the amplification of high loading of signaling molecules by the framework nanocomposites and the ratiometric strategy, the constructed electrochemical immunosensor exhibits outstanding accuracy, stability, and sensitivity. Under optimal conditions, the detection range spanned from 0.01 U mL−1 to 80 U mL−1, with a calculated limit of detection (LOD) of 0.0089 U mL−1. The immunosensor was successfully applied to real serum sample tests, indicating its potential for clinical detection applications.

Ratiometric electrochemical immunoassay based on 2D Co/Fe MOF decorated with toluidine blue and Fc-labeled Schiff base for accurate assay of alpha-fetoprotein in clinical serum

The high level of alpha-fetoprotein (AFP) expression is closely related to hepatocellular carcinoma (HCC). Herein, a dual signal ratiometric electrochemical immunosensor based on chitosan-ferrocenecarboxaldehyde-spindle gold (Chit-Fc-SAu) and Co/Fe metal-organic framework-toluidine blue/polydopamine (Co/Fe MOF-TB/PDA) was proposed for quantitative analysis of AFP. Specifically, Chit-Fc-SAu worked as a substrate to trap more primary antibodies (Ab1) generating the first electrochemical signal from Fc. Thanks to the large specific surface area, the synergistic and electronic effects of Co/Fe MOF nanosheets, and the rich functional groups of PDA, Co/Fe MOF-TB/PDA could load more secondary antibodies (Ab2) and signal molecules (TB) providing another amplified electrochemical signal. In the presence of AFP, Ab1-AFP-Ab2 formed a sandwich structure, and as the AFP concentration increased, the peak current ratio of TB to Fc (ITB/IFc) also increased. The dual signal ratiometric strategy can avoid environmental signal interference and achieve signal self-calibration, thereby improving the accuracy and reproducibility of detection. After a series of exploration, this self-calibrated ratiometric immunosensor exhibited a wide linear range (0.001−200 ng mL−1), a low detection limit (0.34 pg mL−1), and good repeatability. When applied to the assay of clinical serum samples, the detection results of ratiometric sensor were consistent with that of commercial electrochemiluminescence (ECL) immunoassay, significantly superior to that of non-ratiometric sensor. The self-calibrated strategy based on ratiometric sensor helps to improve the accuracy of AFP in clinical diagnosis.

An electrochemical ratiometric immunosensor for the detection of NMP22 based on ZIF-8@MWCNTs@Chit@Fc@AuNPs and AuPt-MB.

Nuclear matrix protein 22 (NMP22) is one of the most important tumor markers of bladder cancer and is significantly elevated in the urine of bladder cancer patients. Therefore, in this work, a highly sensitive ratiometric electrochemical immunosensor was constructed to detect NMP22 based on ZIF-8@MWCNTs@Chit@Fc@AuNPs composites. ZIF-8 had a large surface area and good adsorption ability. Multi-Walled Carbon Nanotubes (MWCNTs) can optimize the electrical conductivity of ZIF-8, so that the electrode surface of ferrocene (Fc) obtains a stable and strong electrochemical signal. In addition, AuPt-MB provided another strong detection signal methylene blue (MB) while immobilizing the secondary antibody (Ab2) through Au-N and Pt-N bonds. A ratiometric electrochemical sensor was formed based on ZIF-8@MWCNTs@Chit@Fc@AuNPs and AuPt-MB, which showed a great linear connection between IMB/IFc and the logarithmic concentration of NMP22 with a detection limit of 3.33 fg mL-1 (S/N = 3) under optimized specifications in the concentration interval of 0.01 pg mL-1 to 1000 ng mL-1. In addition, the ratiometric immunosensor showed good selectivity and stability.

Electrochemical ratiometric dual-signal immunoassay for accurate detection of carcinoembryonic antigen in clinical serum based on rGO-Pd@Au-Thi and Chi-Fc-Au

For prevention and early diagnosis of carcinoembryonic antigen (CEA), it is necessary to develop more accurate approaches for its detection. In this work, a new sensitive electrochemical ratiometric immunosensor for the determination of CEA was constructed on account of chitosan-ferrocenecarboxaldehyde-gold nanospheres (Chi-Fc-Au) and reduced graphene oxide-Pd@Au nanocomposits-thionine (rGO-Pd@Au-Thi) with dual-signal output. The immunosensor utilized Chi-Fc-Au as the substrate to anchor the primary antibody (Ab1) and rGO-Pd@Au-Thi as the secondary antibody (Ab2) label probe, generating two electrical signals (IFc and IThi) derived from Fc and Thi in the presence of CEA, respectively. With the increase of CEA concentration, the IFc and IThi showed opposite changes, so the peak current ratio of IThi/IFc was positively correlated with CEA concentration. In comparison with other immunosensors especially single-signal mode, the proposed immunosensor exhibited a wider linear range (10–4−200 ng mL−1) and a lower detection limit (14.9 fg mL−1), which could be applied in human serum samples for CEA measurement, and was comparable to commercial electrochemiluminescence (ECL) immunoassay. Given the significantly improved accuracy of the constructed sensor, it is expected to be applied in clinical diagnosis for other protein biomarkers determination.

Particle Size-Controlled Oxygen Reduction and Evolution Reaction Nanocatalysts Regulate Ru(bpy)32+'s Dual-potential Electrochemiluminescence for Sandwich Immunoassay.

Multiple signal strategies remarkably improve the accuracy and efficiency of electrochemiluminescence (ECL) immunoassays, but the lack of potential-resolved luminophore pairs and chemical cross talk hinders their development. In this study, we synthesized a series of gold nanoparticles (AuNPs)/reduced graphene oxide (Au/rGO) composites as adjustable oxygen reduction reaction and oxygen evolution reaction catalysts to promote and modulate tris(2,2'-bipyridine) ruthenium(II) (Ru(bpy)32+)'s multisignal luminescence. With the increase in the diameter of AuNPs (3 to 30 nm), their ability to promote Ru(bpy)32+'s anodic ECL was first impaired and then strengthened, and cathodic ECL was first enhanced and then weakened. Au/rGOs with medium-small and medium-large AuNP diameters remarkably increased Ru(bpy)32+'s cathodic and anodic luminescence, respectively. Notably, the stimulation effects of Au/rGOs were superior to those of most existing Ru(bpy)32+ co-reactants. Moreover, we proposed a novel ratiometric immunosensor construction strategy using Ru(bpy)32+'s luminescence promoter rather than luminophores as tags of antibodies to achieve signal resolution. This method avoids signal cross talk between luminophores and their respective co-reactants, which achieved a good linear range of 10-7 to 10-1 ng/ml and a limit of detection of 0.33 fg/ml for detecting carcinoembryonic antigen. This study addresses the previous scarcity of the macromolecular co-reactants of Ru(bpy)32+, broadening its application in biomaterial detection. Furthermore, the systematic clarification of the detailed mechanisms for converting the potential-resolved luminescence of Ru(bpy)32+ could facilitate an in-depth understanding of the ECL process and should inspire new designs of Ru(bpy)32+ luminescence enhancers or applications of Au/rGOs to other luminophores. This work removes some impediments to the development of multisignal ECL biodetection systems and provides vitality into their widespread applications.

Dual-wavelength ratiometric immunosensor for Bacillus cereus: Oxidase-like MnO2-Au trigged “OFF-ON” detection strategy

As an opportunistic pathogen, Bacillus cereus (B. cereus) has caused increasing emetic and diarrheal food poisoning, even fulminant hepatic failure and death case. Herein, a mimetic oxidase-mediated ratiometric fluorescence enzyme-linked immunosorbent assay (ELISA) was developed to detect B. cereus. In this system, 3D MnO2-Au nanoflowers with layered crystalline structure can oxidize o-phenylenediamine (OPD) to generate a fluorescence peak at 570 nm (I570, response signal), while blue carbon quantum dots (bCDs) display a fluorescence peak at 455 nm (I455, reference signal). Under the effect of fluorescence resonance energy transfer (FRET), the response signal increased while the reference signal was weakened. By using MnO2-Au as the mimetic oxidase to replace the horseradish peroxidase and associate with antibody in traditional ELISA, a linear relationship was established between the ratiometric fluorescence signal (I570/I455) and the logarithm of B. cereus concentrations in the range from 1.7 × 102 to 3.3 × 106 CFU/mL. Comparing with conventional sandwich ELISA, the detection time was shortened by 43 min and the sensitivity was improved by 5-folds, indicating that the proposed ratiometric immunoassay holds great potential as a rapid and convenient tool for sensitive screening of B. cereus.

Ratiometric immunosensor with DNA tetrahedron nanostructure as high-performance carrier of reference signal and its applications in selective phoxim determination for vegetables

A ratiometric electrochemical immunosensor, based on DNA tetrahedron nanostructure (DTNS), is introduced for vegetable phoxim determination. DTNS spontaneously adheres onto gold-nanoparticle-modified electrode and forms stable three-dimensional structure, providing plenty of binding sites to the built-in reference, methylene blue (MB). Monoclonal antibody (m-Ab) is vertically linked onto DTNS vertex, selectively responses antigenic phoxim, and promotes the target signal of IPHO. Thus, a ratiometric indicator, IPHO/IMB, is sensibly established with the target signal (IPHO) and the reference signal (IMB). Modifications, mechanisms and advances of the proposed method are subsequently examined with morphological methods and electrochemical experiments. This method brings considerable advances in analytical behaviors. The ratiometric signal presents better performance than solo system in repeatability and long-time stability. As-fabricated sensor presents wide dynamic range as 0.1∼30 μg/L, and limit of detection is well defined as 0.003 μg/L (S/N = 3). Finally, this method is verified with real-vegetable-sample analysis, certified HPLC and recovery test.

AuPt nanocrystals/polydopamine supported on open-pored hollow carbon nanospheres for a dual-signaling electrochemical ratiometric immunosensor towards h-FABP detection

For early diagnosis and prevention of acute myocardial infarction (AMI), accurate and sensitive analysis of cardiac biomarkers by a single test has faced substantial challenge. A novel electrochemical immuno-sensing platform was built for untrasensitive assay of heart-type fatty acid-binding protein (h-FABP) via multi-signal output. This immunosensor utilized Au nanodendrites/chitosan-grafted-ferrocene (Au NDs/Chit-g-Fc) as the substrate and thionine-absorbed AuPt nanocrystals/polydopamine/open-pored hollow carbon spheres (Thi/AuPt/PDA/OHCSs) as the label material, producing two independent electrochemical signals from the Fc and Thi probes, respectively. For such assay of the target h-FABP in the immuno-sensing system, the ratios (IThi / IFc) between the double signals showed a broad dynamic range of 0.001–200.0 ng mL−1 and a low detection limit of 0.53 pg mL−1 (S/N = 3). This ratiometric sensor exerts a far-reaching influence on other biomarkers researches in practical applications.

A label-free three potential ratiometric electrochemiluminescence immunosensor for cardiac troponin I based on N-(4-aminobutyl)-N-ethylisoluminol functionalized graphene quantum dots

• An ECL nanoluminophore with three potential-resolved emissions was synthesized. • A label-free three potential ratiometric immunosensor was built. • A low detection limit of 0.35 fg/mL was obtained for cardiac troponin I. • The developed strategy can effectively eliminate errors caused by environmental changes. In this work, we synthesized a novel ECL nanoluminophore N-(4-aminobutyl)-N-ethylisoluminol functionalized graphene quantum dots (ABEI@GQDs), which exhibited three completely potential-resolved dual-color ECL emissions with coreactant K 2 S 2 O 8 in an aqueous solution. Blue color ECL-1, orange red color ECL-2 and blue color ECL-3 were observed at peak potential of -0.58, -1.85 and 1.20 V, respectively. On this basis, a label-free three potential ratiometric ECL immunosensor was developed for detecting cardiac troponin I (cTnI) by layer-by-layer assembly of ABEI@GQDs and antibody on a chitosan modified fluorine doped tin oxide electrode. In the presence of cTnI, ECL-2 increased, whereas ECL-1 and ECL-3 changed slightly and synchronously. It was found that ECL-1/ECL-3 ratio almost unchanged with increasing the concentration of cTnI, which laid the foundation for self-correction analysis. Accordingly, three potential ratiometric strategy based on the ratio of three ECL peaks was established for the determination of cTnI with a linear dynamic range from 1.0 fg/mL to 5.0 pg/mL and a detection limit of 0.35 fg/mL, which was superior to most reported ECL methods. Moreover, the developed three potential ratiometric ECL immunosensor can effectively eliminate systematic errors caused by environmental changes and thus offers more precise detection results than two potential ratiometric ECL immonosensors.

(Invited) Study of Electrochemiluminescence Nanohybrid and Their Application in Biomarker Detection of Myocardial Infarction

Electrochemiluminescence (ECL) as an analytical technique has attracted increasing attention in clinical diagnosis and environmental assays due to the controllability in temporal, space, and light emitting intensity, simple operation, cost-effectiveness, high sensitivity, and low background signal. Compared to other methods for biomarker detection of Myocardial Infarction, ECL shows high sensitivity and a wide detecting range.The common biomarkers for myocardial infarction include troponin, creatine kinase, myoglobin copeptin, and miRNA. Among those biomarkers, cTnI not only has excellent cardiac specificity but also can provide a long detecting window for cardiac injury, which has been acknowledged as the principal biomarker for AMI.We have developed ECL nanohybrid materials and detection methods for the applications in biomarker detection of myocardial infarction, which include Ru-MOF nanosheet, Ru linking BPEI-coated carbon dots, dual-wavelength ratiometric ECL biosensor, ratiometric ECL–electrochemical(EC) hybrid biosensor, wavelength-resolved ECL resonance energy transfer (ECL-RET) ratiometric immunosensor and so on. These ECL biosensors immunosensors exhibit excellent sensitivity, specificity, stability, and reproducibility. Therefore, they have great application prospects for biomarker detection in clinical diagnosis.ACKNOWLEDGMENTSThis work was supported by the National Key Research and Development Program of China (2016YFA0201301), the National Natural Science Foundation of China (No. 21435005 and 21627808), the Key Research Program of Frontier Sciences, CAS (no. QYZDY-SSW-SLH019).

A self-enhanced electrochemiluminescent ratiometric zearalenone immunoassay based on the use of helical carbon nanotubes.

A self-enhanced electrochemiluminescent ratiometric immunoassay for zearalenone is described. A system composed of N-aminobutyl-N-ethylisoluminol (ABEI) and glutathione (GSH) produces a strong electrochemiluminescence (ECL) at an applied potential of 0.8V, probably because of short electron transfer distance and reduced energy loss. The method also uses octahedral anatase mesocrystals (OAM) with a large specific surface facilitating immobilization of ABEI and GSH. Helical carbon nanotubes, possessing a large specific surface, superior mechanical stability, and excellent electrical conductivity which serve as a solid support, greatly enhanced the loading capacity for g-C3N4 nanosheets and horseradish peroxidase-labeled anti-antibody. The peroxidase accelerates the decomposition of H2O2 to produce reactive oxygen species (ROSs), amplifying the blue ECL of ABEI and the green ECL of g-C3N4. The ratiometric sandwich immunoassay (performed by the ratio of ECL intensity at - 1.3V and 0.8V) allows for sensitive and reliable determination of ZEN in awide linear range from 1.0 × 10-4ng/mL to 10ng/mL. The method was successfully applied to the analysis of corn hazelnut samples for ZEN. Graphical abstract Schematic presentation of a self-enhanced electrochemiluminescent ratiometric immunosensor based on octahedral anatase mesocrystals (OAM) supported ABEI-glutathione (GSH) and g-C3N4 functionalized helical carbon nanotubes (HCNT) for zearalenone (ZEN) determination.

Dual-function of ZnS/Ag2S nanocages in ratiometric immunosensors for the discriminant analysis of ochratoxins: Photoelectrochemistry and electrochemistry

A ratiometric immunosensor was built to detect different ochratoxins in a mixture by combining broad-specific antibody and antigen, multidimensional signals, and computer programs. This immunosensor used MoS2 NFs/CdS as the photoelectrode to immobilize antigens and ZnS/Ag2S nanocages to capture the secondary antibody. During the immunoreaction recognition, the photoelectrochemical (PEC) current of MoS2 NFs/CdS and the square wave voltammetric (SWV) current of the dissolved Zn2+ from ZnS can be influenced by the deposition of the ZnS/Ag2S label. The results show that the ratio of PEC to SWV current decreased linearly with the logarithm of ochratoxin concentrations in a range from 1 ng/L to 1 μg/L. The present work provides a possible route to design broad-specific immunosensors and expands the scope of ratiometric biosensors in the detection of a mixture.

Ratiometric Immunosensor for GP73 Detection Based on the Ratios of Electrochemiluminescence and Electrochemical Signal Using DNA Tetrahedral Nanostructure as the Carrier of Stable Reference Signal.

DNA tetrahedron nanostructure (DTNs) has the merits of simple synthesis, high yield, structural stability, and mechanical rigidity, and its three-dimensional structure provides a satisfactory biosensing interface to the improvement of the binding efficiency of antigenic proteins and antibodies. Electrochemiluminescence (ECL) reagent, tris(4,4'-dicarboxylicacid-2,2'-bipyridyl)ruthenium(II) dichloride (Ru(dcbpy)3Cl2), was modified on the electrode through the formation of classical sandwich complex of antibody-antigen-antibody. ECL response of the system increased with the increment of the target (golgi protein 73 (GP73) in this study) with high selectivity. Besides, the composed double-stranded DNA (dsDNA) in each side of DTNs could act as an excellent carrier of methylene blue (MB), thus producing a stable electrochemical internal reference signal on the electrode surface to correct the potential interferences. Therefore, a highly selective and reproductive ratiometric immunosensor was developed on the basis of the ratio of ECL of Ru(dcbpy)3Cl2 and electrochemistry of MB. The ratio value of the ECL/electrochemistry had a linear relationship with GP73 concentration in the range of 15 pg/mL-0.7 ng/mL, and the limit of detection was 15 pg/mL. The proposed ratiometric ECL immunoassay has been applied to detect GP73 in real serum samples with satisfactory results.

A wavelength-resolved electrochemiluminescence resonance energy transfer ratiometric immunosensor for detection of cardiac troponin I.

In this study, a wavelength-resolved electrochemiluminescence resonance energy transfer (ECL-RET) ratiometric immunosensor from Au nanoparticle functionalized graphite-like carbon nitride nanosheets (Au-g-C3N4) to Au nanoclusters (Au NCs) has been constructed for the first time. At a working voltage of 0 to -1.2 V, Au-g-C3N4 showed a strong cathodic ECL emission with a peak at 460 nm, which overlapped well with the absorption spectra of Au NCs thus stimulating the fluorescence emission of Au NCs at 610 nm. Moreover, within this voltage range, the Au NCs showed no ECL signal; therefore, they would not interfere with the detection of the system. We used cardiac troponin I (cTnI) as an analytical model to construct a sandwich immunosensor based on the ECL-RET ratiometric strategy. By measuring the responses of the ECL460 nm/FL610 nm ratio at different cTnI concentrations, the sensitive detection of cTnI with a wide range of 50 fg mL-1 to 50 ng mL-1 and a low detection limit of 9.73 fg mL-1 can be achieved. This work enriches the wavelength-resolved ECL-RET system and provides an innovative reference for the development of more efficient and sensitive ECL-RET ratiometry.