Recent advance of RNA aptamers and DNAzymes for MicroRNA detection.

MicroRNAs (miRNAs) are a class of small, single-stranded, and non-coding RNA molecules that act as post-transcriptional regulators of gene expression, participating in the regulation of a variety of important biological activities. Accumulating evidence suggests that miRNAs are closely related to many major human diseases, especially cancer, and they are considered to be highly promising diagnostic biomarkers and therapeutic targets for disease diagnosis and treatment. To this end, the development of highly accurate, selective, and sensitive strategies for miRNA detection is essential for realizing the early diagnosis of diseases and improving the success rate of treatment. Over the past decade, functional nucleic acid nanostructures have emerged as powerful tools for detecting disease-related miRNAs because of their unique advantages, e.g., high stability, specificity, and activity. Particularly, thanks to the rapid advancement of systematic evolution of ligands by exponential enrichment (SELEX) technology, it is now feasible to strictly select and reasonably design functional nucleic acids with high specificity and activity toward targets of interest, and thereby enhance the performance of miRNA detection. In this article, we present a comprehensive review of the application of functional nucleic acids including RNA aptamers and DNAzymes selected by SELEX in the construction of biosensors for miRNA detection in recent years. We also provide insights into the impact of the advantages of RNA aptamers and DNAzymes on the enhancement of the performance of miRNA biosensors. We hope this review will serve as a valuable foundation to inspire more exciting research in this emerging field in near future.

Pd@Pt Nanodendrites as Peroxidase Nanomimics for Enhanced Colorimetric ELISA of Cytokines with Femtomolar Sensitivity.

Colorimetric enzyme-linked immunosorbent assay (ELISA) has been widely applied as the gold-standard method for cytokine detection over decades. However, it has become a critical challenge to further improve the detection sensitivity of ELISA as limited by the catalytic activity of enzymes. Herein, we report an enhanced colorimetric ELISA for ultrasensitive detection of interleukin-6 (IL-6, as a model cytokine for demonstration) using Pd@Pt core@shell nanodendrites (Pd@Pt NDs) as peroxidase nanomimics (named "Pd@Pt ND ELISA"), pushing the sensitivity up to femtomolar level. Specifically, the Pd@Pt NDs are rationally engineered by depositing Pt atoms on Pd nanocubes (NCs) to generate rough dendrite-like Pt skins on the Pd surfaces via Volmer-Weber growth mode. They can be produced on a large scale with highly uniform size, shape, composition, and structure. They exhibit significantly enhanced peroxidase-like catalytic activity with catalytic constants (Kcat) more than 2000-fold higher than those of horseradish peroxidase (HRP, an enzyme commonly used in ELISA). Using Pd@Pt NDs as the signal labels, the Pd@Pt ND ELISA presents strong colorimetric signals for the quantitative determination of IL-6 with a wide dynamic range of 0.05-100 pg mL-1 and an ultralow detection limit of 0.044 pg mL-1 (1.7 fM). This detection limit is 21-fold lower than that of conventional HRP-based ELISA. The reproducibility and specificity of the Pd@Pt ND ELISA are excellent. More significantly, the Pd@Pt ND ELISA was validated for analyzing IL-6 in human serum samples with high accuracy and reliability through recovery tests. Our results demonstrate that the colorimetric Pd@Pt ND ELISA is a promising biosensing tool for ultrasensitive determination of cytokines and thus is expected to be applied in a variety of clinical diagnoses and fundamental biomedical studies.

Machine-Learning-Assisted Microfluidic Nanoplasmonic Digital Immunoassay for Cytokine Storm Profiling in COVID-19 Patients.

Cytokine storm, known as an exaggerated hyperactive immune response characterized by elevated release of cytokines, has been described as a feature associated with life-threatening complications in COVID-19 patients. A critical evaluation of a cytokine storm and its mechanistic linkage to COVID-19 requires innovative immunoassay technology capable of rapid, sensitive, selective detection of multiple cytokines across a wide dynamic range at high-throughput. In this study, we report a machine-learning-assisted microfluidic nanoplasmonic digital immunoassay to meet the rising demand for cytokine storm monitoring in COVID-19 patients. Specifically, the assay was carried out using a facile one-step sandwich immunoassay format with three notable features: (i) a microfluidic microarray patterning technique for high-throughput, multiantibody-arrayed biosensing chip fabrication; (ii) an ultrasensitive nanoplasmonic digital imaging technology utilizing 100 nm silver nanocubes (AgNCs) for signal transduction; (iii) a rapid and accurate machine-learning-based image processing method for digital signal analysis. The developed immunoassay allows simultaneous detection of six cytokines in a single run with wide working ranges of 1-10,000 pg mL-1 and ultralow detection limits down to 0.46-1.36 pg mL-1 using a minimum of 3 µL serum samples. The whole chip can afford a 6-plex assay of 8 different samples with 6 repeats in each sample for a total of 288 sensing spots in less than 100 min. The image processing method enhanced by convolutional neural network (CNN) dramatically shortens the processing time ∼6,000 fold with a much simpler procedure while maintaining high statistical accuracy compared to the conventional manual counting approach. The immunoassay was validated by the gold-standard enzyme-linked immunosorbent assay (ELISA) and utilized for serum cytokine profiling of COVID-19 positive patients. Our results demonstrate the nanoplasmonic digital immunoassay as a promising practical tool for comprehensive characterization of cytokine storm in patients that holds great promise as an intelligent immunoassay for next generation immune monitoring.

Nanoplasmonic Sandwich Immunoassay for Tumor-Derived Exosome Detection and Exosomal PD-L1 Profiling.

Tumor-derived exosomes play a vital role in the process of cancer development. Quantitative analysis of exosomes and exosome-shuttled proteins would be of immense value in understanding cancer progression and generating reliable predictive biomarkers for cancer diagnosis and treatment. Recent studies have indicated the critical role of exosomal programmed death ligand 1 (PD-L1) in immune checkpoint therapy and its application as a patient stratification biomarker in cancer immunotherapy. Here, we present a nanoplasmonic exosome immunoassay utilizing gold-silver (Au@Ag) core-shell nanobipyramids and gold nanorods, which form sandwich immune complexes with target exosomes. The immunoassay generates a distinct plasmonic signal pattern unique to exosomes with specific exosomal PD-L1 expression, allowing rapid, highly sensitive exosome detection and accurate identification of PD-L1 exosome subtypes in a single assay. The developed nanoplasmonic sandwich immunoassay provides a novel and viable approach for tumor cell-derived exosome detection and analysis with quantitative molecular details of key exosomal proteins, manifesting its great potential as a transformative diagnostic tool for early cancer detection, prognosis, and post-treatment monitoring.

Morphology-Invariant Metallic Nanoparticles with Tunable Plasmonic Properties.

Current methods for tuning the plasmonic properties of metallic nanoparticles typically rely on alternating the morphology (i.e., size and/or shape) of nanoparticles. The variation of morphology of plasmonic nanoparticles oftentimes impairs their performance in certain applications. In this study, we report an effective approach based on the control of internal structure to engineer morphology-invariant nanoparticles with tunable plasmonic properties. Specifically, these nanoparticles were prepared through selective growth of Ag on the inner surfaces of preformed Ag-Au alloyed nanocages as the seeds to form Ag@(Ag-Au) shell@shell nanocages. Plasmonic properties of the Ag@(Ag-Au) nanocages can be conveniently and effectively tuned by varying the amount of Ag deposited on the inner surfaces, during which the overall morphology of the nanocages remains unchanged. To demonstrate the potential applications of the Ag@(Ag-Au) nanocages, they were applied to colorimetric sensing of human carcinoembryonic antigen (CEA) that achieved low detection limits. This work provides a meaningful concept to design and craft plasmonic nanoparticles.

Template Regeneration in Galvanic Replacement: A Route to Highly Diverse Hollow Nanostructures.

The ability to produce a diverse spectrum of hollow nanostructures is central to the advances in many current and emerging areas of technology. Herein, we report a general method to craft hollow nanostructures with highly tunable physical and chemical parameters. The key strategy is to regenerate the nanoscale sacrificial templates in a galvanic replacement reaction through site-selective overgrowth. As examples, we demonstrate the syntheses of nanocages and nanotubes made of silver, gold, palladium, and/or platinum with well-controlled wall thicknesses and elemental distributions. Using the nanocages of silver and gold as models, we demonstrate they possess intriguing plasmonic properties and offer superior performance in biosensing applications. This study provides a powerful platform to customize hollow nanostructures with desired properties and therefore is expected to enable a variety of fundamental studies and technologically important applications.

Strain Effect in Palladium Nanostructures as Nanozymes.

While various effects of physicochemical parameters (e.g., size, facet, composition, and internal structure) on the catalytic efficiency of nanozymes (i.e., nanoscale enzyme mimics) have been studied, the strain effect has never been reported and understood before. Herein, we demonstrate the strain effect in nanozymes by using Pd octahedra and icosahedra with peroxidase-like activities as a model system. Strained Pd icosahedra were found to display 2-fold higher peroxidase-like catalytic efficiency than unstrained Pd octahedra. Theoretical analysis suggests that tensile strain is more beneficial to OH radical (a key intermediate for the catalysis) generation than compressive strain. Pd icosahedra are more active than Pd octahedra because icosahedra amplify the surface strain field. As a proof-of-concept demonstration, the strained Pd icosahedra were applied to an immunoassay of biomarkers, outperforming both unstrained Pd octahedra and natural peroxidases. The findings in this research may serve as a strong foundation to guide the design of high-performance nanozymes.

Platinum-Decorated Gold Nanoparticles with Dual Functionalities for Ultrasensitive Colorimetric in Vitro Diagnostics.

Au nanoparticles (AuNPs) as signal reporters have been utilized in colorimetric in vitro diagnostics (IVDs) for decades. Nevertheless, it remains a grand challenge to substantially enhance the detection sensitivity of AuNP-based IVDs as confined by the inherent plasmonics of AuNPs. In this work, we circumvent this confinement by developing unique dual-functional AuNPs that were engineered by coating conventional AuNPs with ultrathin Pt skins of sub-10 atomic layers (i.e., Au@Pt NPs). The Au@Pt NPs retain the plasmonic activity of initial AuNPs while possessing ultrahigh catalytic activity enabled by Pt skins. Such dual functionalities, plasmonics and catalysis, offer two different detection alternatives: one produced just by the color from plasmonics (low-sensitivity mode) and the second more sensitive color catalyzed from chromogenic substrates (high-sensitivity mode), achieving an "on-demand" tuning of the detection performance. Using lateral flow assay as a model IVD platform and conventional AuNPs as a benchmark, we demonstrate that the Au@Pt NPs could enhance detection sensitivity by 2 orders of magnitude.

A non-enzyme cascade amplification strategy for colorimetric assay of disease biomarkers.

A non-enzyme cascade amplification strategy, based on the dissolution of Ag nanoparticles and a Pt nanocube-catalyzed reaction, for colorimetric assay of disease biomarkers was developed. This strategy overcomes the intrinsic limitations of enzymes involved in conventional enzymatic amplification techniques, thanks to the utilization of noble-metal nanostructures with superior properties.

High-index {hk0} faceted platinum concave nanocubes with enhanced peroxidase-like activity for an ultrasensitive colorimetric immunoassay of the human prostate-specific antigen.

Developing simple, high-efficiency non-enzyme bioassays is of great importance for modern analytical systems, but remains a significant challenge. One promising route is to utilize highly efficient nanocatalysts with the exposure of active crystal facets. Herein, we for the first time propose a novel colorimetric immunoassay for the ultrasensitive detection of the human prostate-specific antigen (PSA) based on using a unique type of nanolabel - high-index {hk0} faceted platinum concave nanocubes (HIF-Pt-CNCs). The proposed HIF-Pt-CNCs exhibit superior peroxidase-like catalytic activity that is ∼1500- and ∼4-fold higher than that of natural horseradish peroxidase and Pt nanospheres, respectively, and thereby can provide powerful signal amplification by catalyzing the oxidation of peroxidase substrates in the presence of hydrogen peroxide. Using the HIF-Pt-CNC-labelled anti-PSA detection antibody as a signal probe, the immunoassay is carried out in anti-PSA capture antibody-immobilized microplate wells in a sandwich-type detection mode. Under optimal conditions, the developed immunoassay is able to achieve high sensitivity and specificity for PSA detection in a linear range of 20-2000 pg mL-1 and with an ultralow detection limit of 0.8 pg mL-1, which is much lower than that of conventional enzyme-linked immunosorbent assay (ELISA). Moreover, the method is validated for the analysis of 10 PSA clinical serum specimens, and the results agree very well with those obtained by using a commercialized ELISA kit. Therefore, this new, facile and efficient immunoassay is a promising technique with potential applications in medical science research and clinical diagnosis.

Facile Colorimetric Detection of Silver Ions with Picomolar Sensitivity.

Although various colorimetric methods have been actively developed for the detection of Ag+ ions because of their simplicity and reliability, the limits of detection of these methods are confined to the nanomolar (nM) level. Here, we demonstrate a novel strategy for colorimetric Ag+ detection with picomolar (pM) sensitivity. This strategy involves the use of poly(vinylpyrrolidone)- (PVP-) capped Pt nanocubes as artificial peroxidases that can effectively generate a colored signal by catalyzing the oxidation of peroxidase substrates. In the presence of Ag+ ions, the colored signal generated by these Pt cubes is greatly diminished because of the specific and efficient inhibition of Ag+ toward the peroxidase-like activity of the Pt cubes. This colorimetric method can achieve an ultralow detection limit of 80 pM and a wide dynamic range of 10-2-104 nM. To the best of our knowledge, the method presented in this work shows the highest sensitivity for Ag+ detection among all reported colorimetric methods. Moreover, this method also features simplicity and rapidness as it can be conducted at room temperature, in aqueous solution, and requires only ∼6 min.

Hybridization chain reaction-based colorimetric aptasensor of adenosine 5'-triphosphate on unmodified gold nanoparticles and two label-free hairpin probes.

This work designs a new label-free aptasensor for the colorimetric determination of small molecules (adenosine 5'-triphosphate, ATP) by using visible gold nanoparticles as the signal-generation tags, based on target-triggered hybridization chain reaction (HCR) between two hairpin DNA probes. The assay is carried out referring to the change in the color/absorbance by salt-induced aggregation of gold nanoparticles after the interaction with hairpins, gold nanoparticles and ATP. To construct such an assay system, two hairpin DNA probes with a short single-stranded DNA at the sticky end are utilized for interaction with gold nanoparticles. In the absence of target ATP, the hairpin DNA probes can prevent gold nanoparticles from the salt-induced aggregation through the interaction of the single-stranded DNA at the sticky end with gold nanoparticles. Upon target ATP introduction, the aptamer-based hairpin probe is opened to expose a new sticky end for the strand-displacement reaction with another complementary hairpin, thus resulting in the decreasing single-stranded DNA because of the consumption of hairpins. In this case, gold nanoparticles are uncovered owing to the formation of double-stranded DNA, which causes their aggregation upon addition of the salt, thereby leading to the change in the red-to-blue color. Under the optimal conditions, the HCR-based colorimetric assay presents good visible color or absorbance responses for the determination of target ATP at a concentration as low as 1.0nM. Importantly, the methodology can be further extended to quantitatively or qualitatively monitor other small molecules or biotoxins by changing the sequence of the corresponding aptamer.

Facile Synthesis of Enhanced Fluorescent Gold-Silver Bimetallic Nanocluster and Its Application for Highly Sensitive Detection of Inorganic Pyrophosphatase Activity.

Herein, gold-silver bimetallic nanoclusters (Au-Ag NCs) with the high fluorescent intensity were first synthesized successfully and utilized for the fabrication of sensitive and specific sensing probes toward inorganic pyrophosphatase (PPase) activity with the help of copper ion (Cu(2+)) and inorganic pyrophosphate ion (PPi). Cu(2+) was used as the quencher of fluorescent Au-Ag NC, while PPi was employed as the hydrolytic substrate of PPase. The system consisted of PPi, Cu(2+) ion, and bovine serum albumin (BSA)-stabilized Au-Ag NC. The detection was carried out by enzyme-induced hydrolysis of PPi to liberate copper ion from the Cu(2+)-PPi complex. In the absence of target PPase, free copper ions were initially chelated with inorganic pyrophosphate ions to form the Cu(2+)-PPi complexes via the coordination chemistry, thus preserving the natural fluorescent intensity of the Au-Ag NCs. Upon addition of target PPase into the detection system, the analyte hydrolyzed PPi into phosphate ions and released Cu(2+) ion from the Cu(2+)-PPi complex. The dissociated copper ions readily quenched the fluorescent signal of Au-Ag NCs, thereby resulting in the decrease of fluorescent intensity. Under optimal conditions, the detectable fluorescent intensity of the as-prepared Au-Ag NCs was linearly dependent on the activity of PPase within a dynamic linear range of 0.1-30 mU/mL and allowed the detection at a concentration as low as 0.03 mU/mL at the 3sblank criterion. Good reproducibility (CV < 8.5% for the intra-assay and interassay), high specificity, and long-term stability (90.1% of the initial signal after a storage period of 48 days) were also received by using our system toward target PPase activity. In addition, good results with the inhibition efficiency of sodium fluoride were obtained in the inhibitor screening research of pyrophosphatase. Importantly, this system based on highly enhanced fluorescent Au-Ag NCs offer promise for simple and cost-effective screening of target PPase activity without the needs of sample separation and multiple washing steps.

Terbium ion-coordinated carbon dots for fluorescent aptasensing of adenosine 5'-triphosphate with unmodified gold nanoparticles.

This work reports on a novel time-resolved fluorescent aptasensing platform for the quantitative monitoring of adenosine 5'-triphosphate (ATP) by interaction of dispersive/agglomerate gold nanoparticles (AuNPs) with terbium ion-coordinated carbon dots (Tb-CDs). To construct such a fluorescent nanoprobe, Tb-CDs with high-efficient fluorescent intensity are first synthesized by the microwave method with terbium ions (Tb(3+)). The aptasensing system consists of ATP aptamer, AuNP and Tb-CD. The dispersive/agglomerate gold nanoparticles are acquired through the reaction of the aptamer with target ATP. Upon target ATP introduction, the aptamers bind with the analytes to form new aptamer-ATP complexes and coat on the surface of AuNPs to inhibit their aggregation in the high salt solution. In this case, the fluorescent signal of Tb-CDs is quenched by the dispersive AuNPs on the basis of the fluorescence resonance energy transfer (FRET). At the absence of target analyte, gold nanoparticles tend to aggregate in the high salt state even if the aptamers are present. Thus, the added Tb-CDs maintain their intrinsic fluorescent intensity. Experimental results indicated that the aptasensing system exhibited good fluorescent responses toward ATP in the dynamic range from 40nM to 4.0µM with a detection limit of 8.5nM at 3sblank criterion. The repeatability and intermediate precision is less than 9.5% at three concentrations including 0.04, 0.4 and 2.0µM ATP. The selectivity was acceptable toward guanosine 5'-triphosphate, uridine 5'-triphosphate and cytidine 5'-triphosphate. The methodology was applied to evaluate the blank human serum spiked with target ATP, and the recoveries (at 3 concentration levels) ranged between 97.0% and 103.7%. Importantly, this detection scheme is rapid, simple, cost-effective, and does not require extensive sample preparation or separation.

Label-free hairpin DNA-scaffolded silver nanoclusters for fluorescent detection of Hg²âº using exonuclease III-assisted target recycling amplification.

A new label-free DNA sensing protocol was designed for fluorescent detection of mercury(II) (Hg(2+)), coupling hairpin DNA-scaffolded silver nanocluster (DNA-AgNC) with exonuclease III-assisted target recycling amplification. The assay was carried out through target-induced conformational change of hairpin DNA, while the signal derived from the formed silver nanoclusters on hairpin DNA probes. Initially, target Hg(2+) was specifically coordinated with thymine-thymine (T-T) mismatches to form an intact hairpin DNA. Then, the newly formed hairpin DNA was digested through exonuclease III from blunt 3' termini and restrained at 3' protruding terminus, thus resulting in the release of target Hg(2+) from hairpin DNA. The liberated target Hg(2+) initiated the next cycling, thereby causing the conformational change of numerous hairpin probes from the stem-loop DNA structure to single-stranded DNA. Under the optimal conditions, the fluorescent intensity of the as-produced DNA-AgNCs decreased with the increasing Hg(2+) concentration within a dynamic range from 0.1 nM to 10nM with a detection limit (LOD) of 24 pM. Moreover, the low-cost fluorescent sensing system exhibited high reproducibility and good specificity, thus representing an optional sensing platform for rapid screening of Hg(2+) in environmental water samples.

Target-induced nanocatalyst deactivation facilitated by core@shell nanostructures for signal-amplified headspace-colorimetric assay of dissolved hydrogen sulfide.

Colorimetric assay platforms for dissolved hydrogen sulfide (H2S) have been developed for more than 100 years, but most still suffer from relatively low sensitivity. One promising route out of this predicament relies on the design of efficient signal amplification methods. Herein, we rationally designed an unprecedented H2S-induced deactivation of (gold core)@(ultrathin platinum shell) nanocatalysts (Au@TPt-NCs) as a highly efficient signal amplification method for ultrasensitive headspace-colorimetric assay of dissolved H2S. Upon target introduction, Au@TPt-NCs were deactivated to different degrees dependent on H2S levels, and the degrees could be indicated by using a Au@TPt-NCs-triggered catalytic system as a signal amplifier, thus paving a way for H2S sensing. The combination of experimental studies and density functional theory (DFT) studies revealed that the Au@TPt-NCs with only 2-monolayer equivalents of Pt (θPt = 2) were superior for H2S-induced nanocatalyst deactivation owing to their enhanced peroxidase-like catalytic activity and deactivation efficiency stemmed from the unique synergistic structural/electronic effects between Au nanocores and ultrathin Pt nanoshells. Importantly, our analytical results showed that the designed method was indeed highly sensitive for sensing H2S with a wide linear range of 10-100 nM, a slope of 0.013 in the regression equation, and a low detection limit of 7.5 nM. Also the selectivity, reproducibility, and precision were excellent. Furthermore, the method was validated for the analysis of H2S-spiked real samples, and the recovery in all cases was 91.6-106.7%. With the merits of high sensitivity and selectivity, simplification, low cost, and visual readout with the naked eye, the colorimetric method has the potential to be utilized as an effective detection kit for point-of-care testing.

Hemin/G-quadruplex-based DNAzyme concatamers for in situ amplified impedimetric sensing of copper(II) ion coupling with DNAzyme-catalyzed precipitation strategy.

A new signal-amplification strategy based on copper(II) (Cu(2+))-dependent DNAzyme was developed for sensitive impedimetric biosensing of Cu(2+) in aqueous solution by coupling with target-induced formation of hemin/G-quadruplex-based DNAzyme and enzymatic catalytic precipitation technique. Initially, the target analyte cleaved the Cu(2+)-specific DNAzyme to generate an initiator strand on the sensor. Thereafter, the initiator strand underwent an unbiased strand-displacement reaction between hairpin probes in turn to construct a nicked double-helix, accompanying the formation of hemin/G-quadruplex DNAzyme on the long duplex DNA. The newly formed DNAzyme could oxidize the 4-chloro-1-naphthol (4-CN) to produce an insoluble precipitation on the sensor, thus resulting in a local alteration of the conductivity. Under the optimal conditions, the resistance increased with the increasing Cu(2+) in the sample, and exhibited a wide dynamic working range from 0.1 pM to 5.0 nM with a detection limit of 60 fM. The methodology also displayed a high selectivity for Cu(2+) relative to other potentially interfering ions owing to the highly specific Cu(2+)-dependent DNAzyme, and was applicable for monitoring Cu(2+) in real river samples. Thus, our strategy has a good potential in the environment surveys.

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.

High-resolution colorimetric assay for rapid visual readout of phosphatase activity based on gold/silver core/shell nanorod.

Nanostructure-based visual assay has been developed for determination of enzymatic activity, but most involve in poor visible color resolution and are not suitable for routine utilization. Herein, we designed a high-resolution colorimetric protocol based on gold/silver core/shell nanorod for visual readout of alkaline phosphatase (ALP) activity by using bare-eyes. The method relied on enzymatic reaction-assisted silver deposition on gold nanorod to generate significant color change, which was strongly dependent on ALP activity. Upon target ALP introduction into the substrate, the ascorbic acid 2-phosphate was hydrolyzed to form ascorbic acid, and then, the generated ascorbic acid reduced silver ion to metal silver and coated on the gold nanorod, thereby resulting in the blue shift of longitudinal localized surface plasmon resonance peak of gold nanorod accompanying a perceptible color change from red to orange to yellow to green to cyan to blue and to violet. Under optimal conditions, the designed method exhibited the wide linear range 5-100 mU mL(-1) ALP with a detection limit of 3.3 mU mL(-1). Moreover, it could be used for the semiquantitative detection of ALP from 20 to 500 mU mL(-1) by using the bare-eyes. The coefficients of variation for intra- and interassay were below 3.5% and 6.2%, respectively. Finally, this method was validated for the analysis of real-life serum samples, giving results matched well with those from the 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP)-based standard method. In addition, the system could even be utilized in the enzyme-linked immunosorbent assay (ELISA) to detect IgG at picomol concentration. With the merits of simplification, low cost, user-friendliness, and sensitive readout, the gold nanorod-based colorimetric assay has the potential to be utilized by the public and opens a new horizon for bioassays.

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.