Photocatalytic manipulation of Ca2+ signaling for regulating cellular and animal behaviors via MOF-enabled H2O2 generation.

The in situ generation of H2O2 in cells in response to external stimulation has exceptional advantages in modulating intracellular Ca2+ dynamics, including high controllability and biological safety, but has been rarely explored. Here, we develop photocatalyst-based metal-organic frameworks (DCSA-MOFs) to modulate Ca2+ responses in cells, multicellular spheroids, and organs. By virtue of the efficient photocatalytic oxygen reduction to H2O2 without sacrificial agents, photoexcited DCSA-MOFs can rapidly trigger Ca2+ outflow from the endoplasmic reticulum with single-cell precision in a repeatable and controllable manner, enabling the propagation of intercellular Ca2+ waves (ICW) over long distances in two-dimensional and three-dimensional cell cultures. After photoexcitation, ICWs induced by DCSA-MOFs can activate neural activities in the optical tectum of tadpoles and thighs of spinal frogs, eliciting the corresponding motor behaviors. Our study offers a versatile optical nongenetic modulation technique that enables remote, repeatable, and controlled manipulation of cellular and animal behaviors.

Synergistic Brilliance: Engineered Bacteria and Nanomedicine Unite in Cancer Therapy.

Engineered bacteria are widely used in cancer treatment because live facultative/obligate anaerobes can selectively proliferate at tumor sites and reach hypoxic regions, thereby causing nutritional competition, enhancing immune responses, and producing anticancer microbial agents in situ to suppress tumor growth. Despite the unique advantages of bacteria-based cancer biotherapy, the insufficient treatment efficiency limits its application in the complete ablation of malignant tumors. The combination of nanomedicine and engineered bacteria has attracted increasing attention owing to their striking synergistic effects in cancer treatment. Engineered bacteria that function as natural vehicles can effectively deliver nanomedicines to tumor sites. Moreover, bacteria provide an opportunity to enhance nanomedicines by modulating the TME and producing substrates to support nanomedicine-mediated anticancer reactions. Nanomedicine exhibits excellent optical, magnetic, acoustic, and catalytic properties, and plays an important role in promoting bacteria-mediated biotherapies. The synergistic anticancer effects of engineered bacteria and nanomedicines in cancer therapy are comprehensively summarized in this review. Attention is paid not only to the fabrication of nanobiohybrid composites, but also to the interpromotion mechanism between engineered bacteria and nanomedicine in cancer therapy. Additionally, recent advances in engineered bacteria-synergized multimodal cancer therapies are highlighted.

Recent Advances in Metal-Hydride-Based Disease Treatment.

In comparison to traditional antioxidant treatment methods, the use of hydrogen to eliminate reactive oxygen species from the body has the advantages of high biological safety, strong selectivity, and high clearance rate. As an energy storage material, metal hydrides have been extensively studied and used in transporting hydrogen as clean energy, which can achieve a high hydrogen load and controlled hydrogen release. Considering the antioxidant properties of hydrogen and the delivery ability of metal hydrides, metal-hydride-based disease treatment strategies have attracted widespread attention. Up to now, metal hydrides have been reported for the treatment of tumors and a range of inflammation-related diseases. However, limited by the insufficient investment, the use of metal hydrides in disease treatment still has many shortcomings, such as low targeting efficiency, limited therapeutic activity, and complex material preparation process. Particularly, metal hydrides have been found to have a series of optical, acoustic, and catalytic properties when scaled up to the nanoscale, and these properties are also widely used to promote disease treatment effects. From this new perspective, we comprehensively summarize the very recent research progress on metal-hydride-based disease treatment in this review. Ultimately, the challenges and prospects of such a burgeoning cancer theranostics modality are outlooked to provide inspiration for the further development and clinical translation of metal hydrides.

M13 Bacteriophage-Assisted Recognition and Signal Spatiotemporal Separation Enabling Ultrasensitive Light Scattering Immunoassay.

The demand for the ultrasensitive and rapid quantitative analysis of trace target analytes has become increasingly urgent. However, the sensitivity of traditional immunoassay-based detection methods is limited due to the contradiction between molecular recognition and signal amplification caused by the size effect of nanoprobes. To address this dilemma, we describe versatile M13 phage-assisted immunorecognition and signal transduction spatiotemporal separation that enable ultrasensitive light-scattering immunoassay systems for the quantitative detection of low-abundance target analytes. The newly developed immunoassay strategy combines the M13 phage-assisted light scattering signal fluctuations of gold nanoparticles (AuNPs) with gold in situ growth (GISG) technology. Given the synergy of M13 phage-mediated leverage effect and GISG-amplified light scattering signal modulation, the practical detection capability of this strategy can achieve the ultrasensitive and rapid quantification of ochratoxin A and alpha-fetoprotein in real samples at the subfemtomolar level within 50 min, displaying about 4 orders of magnitude enhancement in sensitivity compared with traditional phage-based ELISA. To further improve the sensitivity of our immunoassay, the biotin-streptavidin amplification scheme is implemented to detect severe acute respiratory syndrome coronavirus 2 spike protein down to the attomolar range. Overall, this study offers a direction for ultrasensitive quantitative detection of target analytes by the synergistic combination of M13 phage-mediated leverage effect and GISG-amplified light scattering signal modulation.

Synthesizing Submicron Polyelectrolyte Capsules to Boost Enzyme Immobilization and Enhance Enzyme-Based Immunoassays.

Polyelectrolyte capsules (PCs) exhibit attractive superiorities in enzyme immobilization, including providing a capacious microenvironment for enzyme conformational freedom, highly effective mass transfer, and protecting enzymes from the external environment. Herein, we provide the first systemic evaluation of submicron PCs (SPCs, 500 nm) for enzyme immobilization. The catalytic kinetics results show that SPC encapsulation affected the affinities of enzymes and substrates but significantly enhanced their catalytic activity. The stability test indicates that SPC-encapsulated horseradish peroxidase (HRP) exhibits ultrahigh resistance to external harsh conditions and has a longer storage life than that of soluble HRP. The proposed encapsulation strategy enables 7.73-, 2.22-, and 11.66-fold relative activities when working at a pH as low as 3, at a NaCl concentration as high as 500 mM, and at a trypsin concentration as high as 10 mg/mL. We find that SPC encapsulation accelerates the cascade reaction efficiency of HRP and glucose oxidase. Owing to SPCs enhancing the catalytic activity of the loaded enzymes, we established an amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Escherichia coli O157:H7 using HRP-loaded SPCs. The detection sensitivity of SPC-improved ELISA was found to be 280 times greater than that of conventional HRP-based ELISA. Altogether, we provide an elaborate evaluation of 500 nm SPCs on enzyme immobilization and its application in the ultrasensitive detection of foodborne pathogens. This evaluation provides evidence to reveal the potential advantage of SPCs on enzyme immobilization for enzyme-based immunoassays. It has excellent biological activity and strong stability and broadens the application prospect in urine, soy sauce, sewage, and other special samples.

Design of disintegrable nanoassemblies to release multiple small-sized nanoparticles.

The therapeutic and diagnostic effects of nanoparticles highly depend on the efficiency of their delivery to targeted tissues, such as tumors. The size of nanoparticles, among other characteristics, plays a crucial role in determining their tissue penetration and retention. Small nanoparticles may penetrate deeper into tumor parenchyma but are poorly retained, whereas large ones are distributed around tumor blood vessels. Thus, compared to smaller individual nanoparticles, assemblies of such nanoparticles due to their larger size are favorable for prolonged blood circulation and enhanced tumor accumulation. Upon reaching the targeted tissues, nanoassemblies may dissociate at the target region and release the smaller nanoparticles, which is beneficial for their distribution at the target site and ultimate clearance. The recent emerging strategy that combines small nanoparticles into larger, biodegradable nanoassemblies has been demonstrated by several groups. This review summarizes a variety of chemical and structural designs for constructing stimuli-responsive disintegrable nanoassemblies as well as their different disassembly routes. These nanoassemblies have been applied as demonstrators in the fields of cancer therapy, antibacterial infection, ischemic stroke recovery, bioimaging, and diagnostics. Finally, we summarize stimuli-responsive mechanisms and their corresponding nanomedicine designing strategies, and discuss potential challenges and barriers towards clinical translation.

"Three-in-One" Multifunctional Nanohybrids with Colorimetric Magnetic Catalytic Activities to Enhance Immunochromatographic Diagnosis.

Colorimetric lateral flow immunoassay (LFIA) with gold nanoparticles (AuNPs) as signal reporters has been widely used in point-of-care testing. Nonetheless, the potential of traditional AuNP-based LFIA for the early diagnosis of disease is often compromised by limited sensitivity due to the insufficient colorimetric signal brightness of AuNPs. Herein, we develop a "three-in-one" multifunctional catalytic colorimetric nanohybrid (Fe3O4@MOF@Pt) composed of Fe3O4 nanoparticles, MIL-100(Fe), and platinum (Pt) nanoparticles. Fe3O4@MOF@Pt displays enhanced colorimetric signal brightness, fast magnetic response, and ultrahigh peroxidase-mimicking activity, which are beneficial to the enhancement of the sensitivity of LFIA by coupling with magnetic separation and catalytic amplification. When integrated with the dual-antibody sandwich LFIA platform, the developed Fe3O4@MOF@Pt can achieve an ultrasensitive immunochromatographic assay of procalcitonin with a sensitivity of 0.5 pg mL-1, which is approximately 2280-fold higher than that of conventional AuNP-based LFIA and superior to previously published immunoassays. Therefore, this work suggests that the proposed catalytic colorimetric nanohybrid can act as promising signal reporters to enable ultrasensitive immunochromatographic disease diagnostics.

Point-of-care COVID-19 diagnostics powered by lateral flow assay.

Since its first discovery in December 2019, the global coronavirus disease 2019 (COVID-19) pandemic caused by the novel coronavirus (SARS-CoV-2) has been posing a serious threat to human life and health. Diagnostic testing is critical for the control and management of the COVID-19 pandemic. In particular, diagnostic testing at the point of care (POC) has been widely accepted as part of the post restriction COVID-19 control strategy. Lateral flow assay (LFA) is a popular POC diagnostic platform that plays an important role in controlling the COVID-19 pandemic in industrialized countries and resource-limited settings. Numerous pioneering studies on the design and development of diverse LFA-based diagnostic technologies for the rapid diagnosis of COVID-19 have been done and reported by researchers. Hundreds of LFA-based diagnostic prototypes have sprung up, some of which have been developed into commercial test kits for the rapid diagnosis of COVID-19. In this review, we summarize the crucial role of rapid diagnostic tests using LFA in targeting SARS-CoV-2-specific RNA, antibodies, antigens, and whole virus. Then, we discuss the design principle and working mechanisms of these available LFA methods, emphasizing their clinical diagnostic efficiency. Ultimately, we elaborate the challenges of current LFA diagnostics for COVID-19 and highlight the need for continuous improvement in rapid diagnostic tests.

Manipulating Intratumoral Fenton Chemistry for Enhanced Chemodynamic and Chemodynamic-Synergized Multimodal Therapy.

Chemodynamic therapy (CDT) uses the tumor microenvironment-assisted intratumoral Fenton reaction for generating highly toxic hydroxyl free radicals (•OH) to achieve selective tumor treatment. However, the limited intratumoral Fenton reaction efficiency restricts the therapeutic efficacy of CDT. Recent years have witnessed the impressive development of various strategies to increase the efficiency of intratumoral Fenton reaction. The introduction of these reinforcement strategies can dramatically improve the treatment efficiency of CDT and further promote the development of enhanced CDT (ECDT)-based multimodal anticancer treatments. In this review, the authors systematically introduce these reinforcement strategies, from their basic working principles, reinforcement mechanisms to their representative clinical applications. Then, ECDT-based multimodal anticancer therapy is discussed, including how to integrate these emerging Fenton reinforcement strategies for accelerating the development of multimodal anticancer therapy, as well as the synergistic mechanisms of ECDT and other treatment methods. Eventually, future direction and challenges of ECDT and ECDT-based multimodal synergistic therapies are elaborated, highlighting the key scientific problems and unsolved technical bottlenecks to facilitate clinical translation.

Recent advances in colorimetry/fluorimetry-based dual-modal sensing technologies.

Tailored to the increasing demands for sensing technologies, the fabrication of dual-modal sensing technologies through combining two signal transduction channels into one method has been proposed and drawn considerable attention. The integration of two sensing signals not only promotes the analytical efficiency with reduced assumption, but also improves the analytical performances with enlarged detection linear range, enhanced accuracy, and boosted application flexibility. The two top-rated output signals for developing dual-modal sensors are colorimetric and fluorescent signals because of their outstanding merits for point of care applications and real-time sensitive sensing. Given the rapid development of material chemistry and nanotechnology, the recent decade has witnessed great advance in colorimetric/fluorimetric signal based dual-modal sensing technologies. The new sensing strategy leads to a broad avenue for various applications in disease diagnosis, environmental monitoring and food safety because of the complementary and synergistic effects of the two output signals. In this state-of-the-art review, we comprehensively summarize different types of colorimetric/fluorimetric dual-modal sensing methods by highlighting representative research in the last 5 years, digging into their sensing methodologies, particularly the working principles of the signal transduction systems. Then, the challenges and future prospects for boosting further development of this research field are discussed.

Development of a rapid and sensitive quantum dot nanobead-based double-antigen sandwich lateral flow immunoassay and its clinical performance for the detection of SARS-CoV-2 total antibodies.

Owing to the over-increasing demands in resisting and managing the coronavirus disease 2019 (COVID-19) pandemic, development of rapid, highly sensitive, accurate, and versatile tools for monitoring total antibody concentrations at the population level has been evolved as an urgent challenge on measuring the fatality rate, tracking the changes in incidence and prevalence, comprehending medical sequelae after recovery, as well as characterizing seroprevalence and vaccine coverage. To this end, herein we prepared highly luminescent quantum dot nanobeads (QBs) by embedding numerous quantum dots into polymer matrix, and then applied it as a signal-amplification label in lateral flow immunoassay (LFIA). After covalently linkage with the expressed recombinant SARS-CoV-2 spike protein (RSSP), the synthesized QBs were used to determine the total antibody levels in sera by virtue of a double-antigen sandwich immunoassay. Under the developed condition, the QB-LFIA can allow the rapid detection of SARS-CoV-2 total antibodies within 15 min with about one order of magnitude improvement in analytical sensitivity compared to conventional gold nanoparticle-based LFIA. In addition, the developed QB-LFIA performed well in clinical study in dynamic monitoring of serum antibody levels in the whole course of SARS-CoV-2 infection. In conclusion, we successfully developed a promising fluorescent immunological sensing tool for characterizing the host immune response to SARS-CoV-2 infection and confirming the acquired immunity to COVID-19 by evaluating the SRAS-CoV-2 total antibody level in the crowd.

Controlled copper in situ growth-amplified lateral flow sensors for sensitive, reliable, and field-deployable infectious disease diagnostics.

A polyethyleneimine (PEI)-assisted copper in-situ growth (CISG) strategy was proposed as a controlled signal amplification strategy to enhance the sensitivity of gold nanoparticle-based lateral flow sensors (AuNP-LFS). The controlled signal amplification is achieved by introducing PEI as a structure-directing agent to regulate the thermodynamics of anisotropic Cu nanoshell growth on the AuNP surface, thus controlling shape and size of the resultant AuNP@Cu core-shell nanostructures and confining free reduction and self-nucleation of Cu2+ for improved reproducibility and decreased false positives. The PEI-CISG-enhanced AuNP-LFS showed ultrahigh sensitivities with the detection limits of 50 fg mL-1 for HIV-1 capsid p24 antigen and 6 CFU mL-1 for Escherichia coli O157:H7. We further demonstrated its clinical diagnostic efficacy by configuring PEI-CISG into a commercial AuNP-LFS detection kit for SARS-CoV-2 antibody detection. Altogether, this work provides a reliable signal amplification platform to dramatically enhance the sensitivity of AuNP-LFS for rapid and accurate diagnostics of various infectious diseases.

Emerging design strategies for constructing multiplex lateral flow test strip sensors.

Conventional lateral flow test strip (LFTS) sensors are insufficiently accurate and reliable due to their single-target detection with limited sample information in a single test. The increasing demand for the simultaneous determination of multiple analytes has recently been accelerating the rapid development of high-throughput and multiplexed LFTS sensing technologies. In this contribution, we systematically summarize the recent achievements on the design, development, and application of multiplexed LFTS sensors for improved rapid on-site diagnostics. The discussion focuses on emerging design strategies to increase multiplexing capacity for enhancing analytical efficiency and precision. As a proof-of-concept, several typical examples are presented. The advantages and disadvantages of such approaches are critically analyzed. Finally, we briefly discuss the current challenges and future perspectives.

Dramatically Enhanced Immunochromatographic Assay Using Cascade Signal Amplification for Ultrasensitive Detection of Escherichia coli O157:H7 in Milk.

The conventional colloidal gold immunochromatographic assay (AuNP-ICA) cannot meet the requirements for the rapid and sensitive detection of Escherichia coli (E. coli) O157:H7 because of its poor sensitivity. Herein, a novel two-step cascade signal amplification strategy that integrates in situ gold growth and nanozyme-mediated catalytic deposition was proposed to enhance the detection sensitivity of conventional AuNP-ICA dramatically. The enhanced strip displayed ultrahigh sensitivity in E. coli O157:H7 detection and had a detection limit of 1.25 × 101 CFU/mL, which is approximately 400-fold lower than that of traditional AuNP-ICA (5 × 103 CFU/mL). The amplified strip had no background signal in the T-line zone in the absence of E. coli O157:H7 even after one round of cascade signal amplification. The enhanced strip demonstrated excellent selectivity against E. coli O157:H7 with a negligible cross-reaction to nine other common pathogens. Intra-assays and interassays showed that the improved strip has acceptable accuracy and precision for determining E. coli O157:H7. The average recoveries in a real milk sample ranged from 87.33 to 112.15%, and the coefficients of variation were less than 10%. The enhanced strip also showed great potential in detecting a single E. coli O157:H7 cell in a 75 µL solution.

Comparison of three sample addition methods in competitive and sandwich colloidal gold immunochromatographic assay.

Immunochromatographic assays (ICAs) are mainstream point-of-care diagnostic tools in disease control, food safety, and environmental monitoring. However, the important issue pertaining to the influence of sample addition methods on the detection performance of ICAs has not been addressed, and related information is still lacking. Herein, we selected the well-accepted gold nanoparticles (AuNPs) as visual labels. AuNP-based ICA was then used to explore the effects of three sample addition methods (i.e., dry, wet, and insert) on the analytical performance of ICAs by using competitive and sandwich models. Under optimized conditions, the competitive ICA with clenbuterol as an analyte showed a negligible difference (p > 0.05) in the detection performance of the three methods in ideal phosphate buffered saline solution. However, the wet method demonstrated the worst performance in pork samples (p < 0.05). The sandwich ICA strip with human chorionic gonadotropin as an analyte revealed the significantly different analytical performances of the three approaches in phosphate buffer (PB) solution and spiked serum (p < 0.05). Two independent linear correlations were observed with the increase in target concentration. However, for the wet method in the PB solution and serum, the first linear correlation was at a relatively narrow target concentration range, and the second linear correlation was at a wider concentration range compared with those for the dry and insert methods. Our findings demonstrated that sample addition methods slightly influence competitive ICAs (p > 0.05) but remarkably affect sandwich ICAs (p < 0.05). We believe that this study can further explain the differences in detection results for the same target analyte in actual ICA detection. The results may serve as a reference in the rational selection of the appropriate sample addition method for succeeding ICA works.

Supramolecular Recognition-Mediated Layer-by-Layer Self-Assembled Gold Nanoparticles for Customized Sensitivity in Paper-Based Strip Nanobiosensors.

Herein, a smart supramolecular self-assembly-mediated signal amplification strategy is developed on a paper-based nanobiosensor to achieve the sensitive and customized detection of biomarkers. The host-guest recognition between ß-cyclodextrin-coated gold nanoparticles (AuNPs) and 1-adamantane acetic acid or tetrakis(4-carboxyphenyl)porphyrin is designed and applied to the layer-by-layer self-assembly of AuNPs at the test area of the strip. Thus, the amplified platform exhibits a high sensitivity with a detection limit at subattogram levels (approximately dozens of molecules per strip) and a wide dynamic range of concentration over seven orders of magnitude. The applicability and universality of this sensitive platform are demonstrated in clinically significant ranges to measure carcinoembryonic antigen and HIV-1 capsid p24 antigen in spiked serum and clinical samples. The customized biomarker detection ability for the on-demand needs of clinicians is further verified through cycle incubation-mediated controllable self-assembly. Collectively, the supramolecular self-assembly amplification method is suitable as a universal point-of-care diagnostic tool and can be readily adapted as a platform technology for the sensitive assay of many different target analytes.

A Gold Growth-Based Plasmonic ELISA for the Sensitive Detection of Fumonisin B1 in Maize.

In this paper, a highly sensitive plasmonic enzyme-linked immunosorbent assay (pELISA) was developed for the naked-eye detection of fumonisin B1 (FB1). Glucose oxidase (GOx) was used as an alternative to horseradish peroxidase as the carrier of the competing antigen. GOx catalyzed the oxidation of glucose to produce hydrogen peroxide, which acted as a reducing agent to reduce Au3+ to Au on the surface of gold seeds (5 nm), This reaction led to a color change in the solution from colorless to purple, which was observable to the naked eye. Various parameters that could influence the detection performance of pELISA were investigated. The developed method exhibited a considerably high sensitivity for FB1 qualitative naked-eye detection, with a visible cut-off limit of 1.25 ng/mL. Moreover, the proposed pELISA showed a good linear range of 0.31-10 ng/mL with a half maximal inhibitory concentration (IC50) of 1.86 ng/mL, which was approximately 13-fold lower than that of a horseradish peroxidase- (HRP)-based conventional ELISA. Meanwhile, the proposed method was highly specific and accurate. In summary, the new pELISA exhibited acceptable accuracy and precision for sensitive naked-eye detection of FB1 in maize samples and can be applied for the detection of other chemical contaminants.

Quantum bead-based fluorescence-linked immunosorbent assay for ultrasensitive detection of aflatoxin M1 in pasteurized milk, yogurt, and milk powder.

Herein, we reported a novel direct competitive fluorescence-linked immunosorbent assay (dcFLISA) for the ultrasensitive detection of aflatoxin M1 (AFM1) in pasteurized milk, yogurt, and milk powder using 150-nm quantum dot beads (QB) as the carrier of competing antigen. Large QB were applied to decrease the binding affinity of the competing antigen to antibody and enhance the fluorescent signal intensity. The aflatoxin B1 molecule was used as the surrogate of AFM1 to label with BSA on the surface of QB because of its 63% cross reaction to anti-AFM1 mAb. The binding affinity of the competing antigen to mAb was tuned by changing the labeled molar ratios of aflatoxin B1 to BSA. Through combining the advantages of QB as the carrier of the competing antigen, including low binding affinity to mAb and highly fluorescent signal output, the proposed dcFLISA exhibited an ultrahigh sensitivity for AFM1 detection, with a half-maximal inhibitory concentration of 3.15 pg/mL in 0.01 M phosphate-buffered saline solution (pH 7.4), which is substantially lower than that of the traditional horseradish peroxidase-based ELISA. The proposed method also exhibited very low detection limitations of 0.5, 0.6, and 0.72 pg/mL for real pasteurized milk, yogurt, and milk powder, respectively. These values are considerably below the maximum permissible level of the European Commission standard for AFM1 in dairy products. In summary, the proposed dcFLISA offers a novel strategy with an ultrahigh sensitivity for the routine monitoring of AFM1 in various dairy products.

Dual-mode fluorescent and colorimetric immunoassay for the ultrasensitive detection of alpha-fetoprotein in serum samples.

We present a novel dual-mode fluorescent and colorimetric immunosensor based on conventional immunoassay platforms by utilizing a gold nanoflower (AuNF)-loaded fluorescein molecule (AuNF@Fluorescein) as signal output. The AuNFs were modified with thiolated carboxyl ligand, which consisted of a hydrophobic alkane chain as hydrophobic wallet for fluorescein encapsulation, a tetra (ethylene glycol) unit for biocompatibility and solubility, and a functional carboxyl group for the conjugation of biorecognition molecules for biosensing. The resultant AuNFs showed a high loading capacity of 3.74 × 106 fluorescein molecules per AuNF because of its flower-like shape with many complex branches. By adjusting the solution pH to 8.0, the fluorescein molecules can almost entirely be released from the hydrophobic wallet of AuNF@Fluorescein, which led to strong fluorescent-signal amplification. Under the optimal detection conditions, the proposed immunoassay based on fluorescent signal exhibited ultrahigh sensitivity for alpha-fetoprotein (AFP) detection, with a limit of detection (LOD) of 29 fg/mL. This value is approximately 9.3 × 103-fold lower than that of corresponding horseradish peroxidase (HRP)-based immunoassay (LOD = 270 pg/mL). The fluorescein molecule also had intrinsic peroxidase-like activity to catalyze 3,3',5,5'-tetramethylbenzidine oxidation with hydrogen peroxide for colorimetric signal. The proposed method with colorimetric mode further exhibited a sensitivity with a LOD of 17.7 pg/mL, which is about 15-fold lower than that of conventional HRP-based immunoassay. The recoveries of the proposed dual-mode immunoassay for AFP spiked serum samples ranged within 89.85%-100.0%, with the coefficient of variations ranging from 0.5% to 2.4%, indicating acceptable accuracy and precision for AFP quantitative detection. The reliability of the developed dual-mode immunoassay was further compared with a commercial chemiluminescence immunoassay kit by analyzing 20 clinical serum samples, showing that the two methods well agreed with each other, with high correlation coefficients of 0.997 and 0.986 based on recorded fluorescence and colorimetric signals, respectively. In summary, the proposed method was highly suitable for the ultrasensitive analysis of biomarkers or infectious diseases by fluorescence mode and can be used for routine clinical diagnosis by colorimetric mode.

Multi-branched gold nanoflower-embedded iron porphyrin for colorimetric immunosensor.

Here, we for the first time used a thiolated amino-ligand modified multi-branched gold nanoflower as skeleton to encapsulate iron porphyrins (AuNF@FeTPPCl) as alternatives to horseradish peroxidase (HRP). After the FeTPPCl encapsulation, the HRP mimicking activity of FeTPPCl was effectively blocked by the steric hindrance of the hydrophobic layer on the AuNF surface. Upon the addition of ethanol, the loaded FeTPPCl was released into the solution in its free format and exposed the catalytic sites, resulting in the recovery of catalytic activity. The proposed encapsulation method effectively avoided the loss of catalytic activity that originated from the blocking of the catalytic active sites during immobilization. Additionally, the resultant AuNF@FeTPPCl nanocomposite exhibited a high loading level with 2.4 × 106 FeTPPCl molecules per AuNF, and showed considerably high catalytic activity for hydrogen peroxide and 3, 3' 5, 5'-tetramethylbenzidine, which is approximately 40- and 172-folds higher than native HRP. Through using the as-prepared AuNF@FeTPPCl as an alternative of HRP for trace labeling, we successfully developed colorimetric immunosensors for fumonisin B1 (FB1) and hepatitis B surface antigen (HBsAg) with competitive and sandwich-type formats, respectively. The developed AuNF@FeTPPCl-based colorimetric immunosensor exhibited higher detection sensitivity for FB1 and HBsAg than the corresponding HRP-based immunosensors. Thus, the proposed AuNF@FeTPPCl can be used as HRP mimicking analogs for developing highly sensitive colorimetric immunosensor and for loading other hydrophobic iron porphyrins or catalysts.