Signal-transduction Tag Research Articles

Dual-recognition colorimetric platform based on porous Au@Pt nanozymes for highly sensitive washing-free detection of Staphylococcus aureus.

Adual-recognition strategy is reportedto construct a one-step washing and highly efficient signal-transduction tag system for high-sensitivity colorimetric detection of Staphylococcus aureus (S. aureus). The porous (gold core)@(platinum shell) nanozymes (Au@PtNEs) as the signal labels show highly efficient peroxidase mimetic activity and arerobust. For the sake of simplicity the detection involved the use of a vancomycin-immobilized magnetic bead (MB) and aptamer-functionalized Au@PtNEs for dual-recognition detection in the presence of S. aureus.In addition, we designed a magnetic plate to fit the 96-well microplate to ensure consistent magnetic properties of each well, which can quickly remove unreacted Au@PtNEs and sample matrix while avoiding tedious washing steps. Subsequently, Au@PtNEs catalyze hydrogen peroxide (H2O2) to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) generating a color signal. Finally, the developed Au@PtNEs-based dual-recognition washing-free colorimetric assay displayed a response in the range of S. aureus of 5 × 101-5 × 105 CFU/mL, and the detection limit was 40 CFU/mL within 1.5 h. In addition, S. aureus-fortified samples were analyzed to further evaluate the performance of the proposed method, which yielded average recoveries ranging from 93.66 to 112.44% and coefficients of variation (CVs) within the range 2.72-9.01%. These results furnish a novel horizon for the exploitation of a different mode of recognition and inexpensive enzyme-free assay platforms as an alternative to traditional enzyme-based immunoassays for the detection of other Gram-positive pathogenic bacteria.

Colorimetric ELISA based on urease catalysis curcumin as a ratiometric indicator for the sensitive determination of aflatoxin B1 in grain products

There is an urgent need to measure aflatoxin B1 (AFB1) in food to prevent contaminated food consumption. In this work, a novel colorimetric enzyme-linked immunosorbent assay (ELISA) was developed for the detection of AFB1 using curcumin as a colorimetric indicator. An indirect competitive enzyme-label immunoassay was developed using urease and rabbit anti-mouse immunoglobulin G labeled with gold nanoparticles as the signal-transduction tag. Urease catalyzed the hydrolysis of urea to produce ammonia, which increased the pH of the solution. The phenolic hydroxyl group of curcumin ionized into phenolic oxygen anions under alkaline conditions, which strengthened the synergistic effect of electron supply and absorption in curcumin. As a result, the color of curcumin changed from yellow to reddish-brown, producing a visible color change. Under optimal conditions, AFB1 could be qualitatively determined with the naked eye, and quantitatively assessed by measuring the ratio of absorbance at wavelengths of 550 and 428 nm. The change in the ratio of absorbance Δ550/Δ428 decreased linearly in a range of 0.01–5 ng mL−1, and the limit of detection was 67 pg mL−1. Therefore, the selectivity and reliability of this proposed method were well validated. This method was also successfully used for the quantitation of AFB1 in spiked rice flour and wheat flour samples. This approach may broaden the application field of colorimetric ELISA for aflatoxin, providing a promising platform for the rapid screening of aflatoxin in food.

Cross-linkage urease nanoparticles: a high-efficiency signal-generation tag for portable pH meter-based electrochemical immunoassay of lipocalin-2 protein diagnostics.

An innovative signal-transduction tag based on cross-linked urease nanoparticles (CLENP) was designed for thedevelopment of apH meter-based immunoassay of lipocalin-2 (LCN2). The CLENP was synthesized with a typical desolvation method using ethanol as desolvation agent, followed by functionalization with polyaspartic acid. The carboxylated CLENP were used as the signal-generation tags for the labelling of secondary antibodies via the carbodiimide coupling. Upon target LCN2 introduction, a sandwich-type immune reaction was performed between capture antibody-coated plate and the labeled secondary antibody on the CLENP. The conjugated CLENP in the microplate hydrolyzed urea into ammonia (NH4+) and carbonate (CO32-), resulting in the pH change of solution, which was determined witha handheld pH meter. The pH variation was proportional to target concentration in the sample. By monitoring thepH variation of theurea solution, the level of LCN2 at a concentration as low as 5.2pgmL-1was evaluated.ThepH meter-based electrochemical immunoassay can be utilized formass production of miniaturized lab-on-a-chip devices with handheld pH meter, thereby opening new opportunities for protein diagnostics and biosecurity. Graphical abstract An innovative signal-transduction tag based on cross-linked urease nanoparticles was designed for high-efficiency immunoassay of lipocalin-2 with pH meter readout.

A fluorescence based immunoassay for galectin-4 using gold nanoclusters and a composite consisting of glucose oxidase and a metal-organic framework

The authors describe a fluorescence immunoassay for galectin-4, a candidate biomarker for various cancers. Glucose oxidase was encapsulated into a zeolitic imidazolate framework to give a composite (GOx/ZIF-8 composite) that acts as a signal-transduction tag via a biomimetic mineralization process. After modification of the composite with streptavidin, it binds biotinylated antibody against galectin-4. In the immunoassay, the response to galectin-4 results from the enzymatic oxidation of glucose. This reaction produces hydrogen peroxide (H2O2) that reacts with iron(II) ions to generate hydroxy radical (•OH), which leads to the quenching of the fluorescence of gold nanoclusters (AuNCs). Accordingly, the fluorescence quenching of AuNCs depends on the concentration of target galectin-4. The GOx/ZIF-8 composite has a high loading capacity for GOx at uncompromised enzymatic activity. The fluorescence of AuNCs is sensitively quenched by •OH radicals. Galectin-4 can be detected by this method in concentrations as low as 10 pg·mL−1. It is expected that this kind of enzyme/MOF composite-based immunoassay has a wide scope in that it may be adapted to other low-abundance proteins and biomarkers.

Fenton reaction-based colorimetric immunoassay for sensitive detection of brevetoxin B

We designed a new colorimetric immunoassay for sensitive monitoring of brevetoxin B (BTB) using enzyme-controlled Fenton reaction with a high-resolution 3,3′,5,5′-tetramethylbenzidine (TMB)-based visual colored system. Upon addition of hydrogen peroxide (H2O2), the equivalent iron(II) could be first converted into iron(III) and free hydroxyl radical (•OH) via the classical Fenton reaction. Then the as-produced iron(III) and •OH could cause a perceptible change from colorless to blue with the increasing H2O2 concentration in the presence of TMB. Based on Fenton reaction-triggered visual colored system, a novel competitive-type colorimetric enzyme immunoassay was developed for the quantitative screening of target BTB on the bovine serum albumin-BTB-modified magnetic bead using glucose oxidase/anti-BTB antibody-labeled gold nanoparticle as the signal-transduction tag. Upon target BTB introduction, the analyte competed with the conjugated BTB on the magnetic bead for anti-BTB antibody on gold nanoparticle. The carried glucose oxidase with the gold nanoparticle could implement the oxidation of glucose to produce H2O2, and the generated H2O2 promoted the above-mentioned Fenton reaction for color development. Under the optimal conditions, the absorbance decreased with the increasing target BTB in the range from 0.1 to 150ngkg−1 with a low detection limit (LOD) of 0.076ngkg−1. The LOD was 500-fold lower than that of commercialized Abraxis BTB ELISA kit. Non-specific adsorption was not observed. The precision, reproducibility and specificity were acceptable. Finally, the method accuracy was also validated for monitoring spiked seafood samples, giving results well matched with the referenced brevetoxin ELISA kit.

Mesoporous carbon-enriched palladium nanostructures with redox activity for enzyme-free electrochemical immunoassay of brevetoxin B

A new signal amplification strategy based on mesoporous carbon-enriched palladium nanostructure (MSC-PdNS) was designed for enzyme-free electrochemical immunoassay of brevetoxin B (BTB) in marine toxins. The assay was carried out on a BTB-bovine serum albumin-functionalized electrode by using monoclonal mouse anti-BTB-labeling MSC-PdNS as the signal-transduction tag. A competitive-type assay protocol was successfully introduced to develop a high-efficiency enzyme-free immunoassay accompanying the doped palladium nanostructure into MSC-PdNS toward reduction of H2O2. Under the optimal conditions, the catalytic current decreased with the increment of BTB concentration in the range from 0.01 to 10 ng mL−1 with a detection limit (LOD) of 5.0 pg mL−1 BTB at the 3sblank criterion. The selectivity and precision were acceptable. In addition, the methodology was further validated for assaying spiked seafood samples, and consistent results between the electrochemical immunoassay and the referenced enzyme immunoassay were obtained. Importantly, the enzyme-free electrochemical immunoassay provides a promising approach for rapid screening of marine toxin because of its simplicity, low cost, sensitivity, specificity and without the need of sample pretreatment.

Urchin-like (gold core)@(platinum shell) nanohybrids: A highly efficient peroxidase-mimetic system for in situ amplified colorimetric immunoassay.

The development of signal-amplified colorimetric immunoassay relies on the design of highly efficient signal-transduction tags. One promising route is to exploit a novel enzyme mimetic system as the signal label. Herein, we report that urchin-like (gold core)@(platinum shell) nanohybrids (Au@PtNHs) can be utilized as a highly efficient peroxidase mimetic system for in situ amplified colorimetric immunoassay of prostate-specific antigen (PSA, one kind of tumor marker). Initially, urchin-like Au@PtNHs were discovered to outperform horseradish peroxidase (HRP) by a vast margin in terms of the turnover number toward hydrogen peroxide (H2O2)-3,3',5,5'-tetramethylbenzidine (TMB) system and the stability against high temperatures and HRP inhibitors. Based on this discovery, the assay was simply carried out on a capture antibody-immobilized microplate by using the Au@PtNH-labeled detection antibody as a signal-transduction tag with a sandwich-type assay mode. The colorimetric signal stemmed from the labeled Au@PtNHs toward catalytic oxidation of TMB-H2O2 system. Experimental results indicated that the Au@PtNH-based colorimetric immunoassay could display a good colorimetric response toward PSA in the dynamic working range of 5-500 pg mL(-1) with a low detection limit of 2.9 pg mL(-1). Meanwhile, the developed immunoassay exhibited good precision and reproducibility, high specificity and acceptable accuracy for the detection of clinical serum samples. These results open up a new horizon for the development of highly sensitive, highly stable and inexpensive non-enzyme immunoassay platforms as an alternative to conventional enzyme-based immunoassay platforms.

HCR-stimulated formation of DNAzyme concatamers on gold nanoparticle for ultrasensitive impedimetric immunoassay

A novel signal-amplified impedimetric immunosensing strategy was successfully developed for ultrasensitive detection of low-abundance proteins (carcinoembryonic antigen, CEA, used as a model) based on hybridization chain reaction (HCR)-stimulated formation of DNAzyme concatamers on nanogold particle accompanying enzyme-triggered biocatalytic precipitation. The assay was carried out on capture antibody-modified electrode by using gold nanoparticle heavily functionalized with initiator strand and detection antibody as the signal-transduction tag with a sandwich-type immunosensing format. In the presence of target CEA, the formed immunocomplex underwent an unbiased strand-displacement reaction by the initiator strand on the gold nanoparticle between two auxiliary single-stranded DNA with the hemin aptamer. Upon hemin introduction, numerous DNAzyme molecules were formed on the concatamers and nanogold particle, which could catalyze 4-chloro-1-naphthol to produce an insoluble precipitation on the electrode, thereby resulting in the amplification of impedimetric signal. Under the optimal conditions, the immuno-HCR assay exhibited good impedimetric responses for the detection of target CEA in the working range from 1.0pgmL−1 to 20ngmL−1 with a detection limit of 0.42pgmL−1. In addition, the immuno-HCR assay was validated by measuring six human serum specimens for target CEA, receiving a highly matched correction between the obtained results by the immuno-HCR assay and the commercialized ELC-based immunoassay method.

Low-cost and highly efficient DNA biosensor for heavy metal ion using specific DNAzyme-modified microplate and portable glucometer-based detection mode

A simple and low-cost DNA sensing platform based on Pb2+-specific DNAzyme-modified microplate was successfully developed for highly sensitive monitoring of lead ion (Pb2+, one kind of toxic heavy metal ion) in the environmental samples coupling with a portable personal glucometer (PGM)-based detection mode. The detection cell was first prepared simply by means of immobilizing the DNAzyme on the streptavidin-modified microplate. Gold nanoparticle labeled with single-stranded DNA and invertase (Enz-AuNP-DNA) was utilized as the signal-transduction tag to produce PGM substrate (glucose). Upon addition of lead ion into the microplate, the substrate strand of the immobilized DNAzyme was catalytically cleaved by target Pb2+, and the newly generated single-strand DNA in the microplate could hybridize again with the single-stranded DNA on the Enz-AuNP-DNA. Accompanying with the Enz-AuNP-DNA, the carried invertase could convert sucrose into glucose. The as-produced glucose could be monitored by using a widely accessible PGM for in situ amplified digital readout. Based on Enz-AuNP-DNA amplification strategy, as low as 1.0pM Pb2+ could be detected under the optimal conditions. Moreover, the methodology also showed good reproducibility and high selectivity toward target Pb2+ against other metal ions because of highly specific Pb2+-dependent DNAzyme, and was applicable for monitoring Pb2+ in the naturally contaminated sewage and spiked drinking water samples.

Biofunctionalized dendritic polyaniline nanofiber for in situ amplified glucometer-based enzyme immunoassay of tumor marker

A new glucometer-based enzyme immunoassay was developed for the sensitive detection of alpha-fetoprotein using biofunctional dendritic polyaniline nanofibers as the signal-transduction tag.

NiCoBP-doped carbon nanotube hybrid: A novel oxidase mimetic system for highly efficient electrochemical immunoassay

NiCoBP-doped multi-walled carbon nanotube (NiCoBP–MWCNT) was first synthesized by using induced electroless-plating method and functionalized with the biomolecules for highly efficient electrochemical immunoassay of prostate-specific antigen (PSA, used as a model analyte). We discovered that the as-synthesized NiCoBP–MWCNT had the ability to catalyze the glucose oxidization with a stable and well-defined redox peak. The catalytic current increased with the increment of the immobilized NiCoBP–MWCNT on the electrode. Transmission electron microscope (TEM) and energy dispersive X-ray spectrometry (EDX) were employed to characterize the as-prepared NiCoBP–MWCNT. Using the NiCoBP–MWCNT-conjugated anti-PSA antibody as the signal-transduction tag, a new enzyme-free electrochemical immunoassay protocol could be designed for the detection of target PSA on the capture antibody-functionalized immunosensing interface. Experimental results revealed that the designed immunoassay system could exhibit good electrochemical responses toward target PSA, and allowed the detection of PSA at a concentration as low as 0.035ngmL−1. More importantly, the NiCoBP-MWCNT-based oxidase mimetic system could be further extended for the monitoring of other low-abundance proteins or disease-related biomarkers by tuning the target antibody.

Tyramine-based enzymatic conjugate repeats for ultrasensitive immunoassay accompanying tyramine signal amplification with enzymatic biocatalytic precipitation.

A new impedimetric immunoassay protocol based on enzyme-triggered formation of tyramine-enzyme repeats on gold nanoparticle (AuNP) was designed for highly sensitive detection of carcinoembryonic antigen (CEA, as a model) by virtue of utilizing enzymatic biocatalytic precipitation toward 4-chloro-1-naphthol (4-CN) on anti-CEA antibody (Ab1)-modified immunosensor. Initially, AuNP was functionalized with horseradish peroxidase and detection antibody (HRP-AuNP-Ab2), and then HRP-tyramine conjugate was utilized for the formation of tyramine-HRP repeats through the triggering of the immobilized HRP on the AuNP with the aid of H2O2. In the presence of target CEA, the carried HRP-tyramine repeats accompanying the sandwiched immunocomplex catalyzed the 4-CN oxidation to produce an insoluble precipitation on the immunosensor, thus causing a local alteration of the conductivity. Three signal-transduction tags including HRP-Ab2, HRP-AuNP-Ab2, and HRP-AuNP-Ab2 with HRP-tyramine repeats were employed for target CEA evaluation, and improved analytical properties were achieved by HRP-AuNP-Ab2 with HRP-tyramine repeats. Using the unique signal-transduction tag, the analytical performance of the impedimetric immunoassay was studied in detail. Under the optimal conditions, the impedimetric immunosensor displayed a wide dynamic working range of between 0.5 pg mL(-1) and 40 ng mL(-1) with a detection limit (LOD) of 0.38 pg mL(-1) relative to target CEA. The coefficients of variation (CVs) were ≤9.3% and 13.3% for the intra-assay and interassay, respectively. The levels of CEA in eight clinical serum specimens were measured by using the developed impedimetric immunosensor. The obtained results correlated well with those from the electrochemiluminescent (ECL)-based immunoassay with a correlation coefficient of 0.998.