Bimetallic alloying gold-silver nanoclusters (Au-AgNCs) have been synthesized by a one-pot biomineralization synthesis route at a vital molar ratio of Au/Ag precursors in the protein matrix. Unexpectedly, the prepared Au-AgNCs could exhibit dramatically enhanced red fluorescence, which is about 6.5-fold and 4.7-fold higher than that of common AuNCs and core-shell Au@AgNCs, respectively. A rapid, selective, and ultrasensitive fluorimetric method has thereby been developed using Au-AgNCs as fluorescent probes toward the separate detections of Hg(2+) and Cu(2+) ions in blood. The interactions of Au-AgNCs with Hg(2+) and Cu(2+) ions were systematically characterized by microscopy imaging, UV-vis, and fluorescence measurements. It is demonstrated that the "silver effect" gives the Au-AgNCs probes not only greatly enhanced red fluorescence but also the strong capacity to specifically sense Cu(2+) ions in addition to improved response to Hg(2+) ions. Moreover, aided by a Cu(2+) chelating agent, exclusive detection of Hg(2+) ions could also be expected with the coexistence of a high level of Cu(2+) ions, as well as reversible Cu(2+) analysis by restoring the fluorescence of Au-AgNCs. Additionally, Au-AgNCs with strong red fluorescence could facilitate fluorimetric analysis with minimal interference from blood backgrounds. Such an Au-AgNCs-based fluorimetric method can allow for the selective analysis of Hg(2+) and Cu(2+) ions down to 0.30 nM and 0.60 nM in blood, respectively, promising a novel detection method to be applied in the clinical laboratory.

Skin interstitial fluid (ISF) containing a great variety of molecular biomarkers derived from cells and subcutaneous blood capillaries has recently emerged as a clinically potential component for early diagnosis of a wide range of diseases; however, the minimally invasive sampling and detection of cell-free biomarkers in ISF is still a key challenge. Herein, we developed microneedles (MNs) that consist of gelatin methacryloyl (GelMA) and graphene oxide (GO) for the enrichment and sensitive detection of multiple microRNA (miRNA) biomarkers from skin ISF. The GO-GelMA MNs exhibited robust mechanical properties, fast sampling kinetics, and large swelling capacity, which enabled collecting ISF volume high to 21.34 μL in 30 min, facilitating effective miRNA analysis. It preliminarily realized the sensitive detection of three types of psoriasis-related miRNAs biomarkers either on the patch itself or in solution after release from the hydrogel by combining catalytic hairpin assembly signal amplification reaction. The automated and minimally invasive ISF miRNA detection technology of GO-GelMA MNs has great potential to monitor cell-free clinically informative biomarkers for personalized diagnosis and prognosis.

Skin interstitial fluid (ISF) is a biofluid with information-rich biomarkers for disease diagnosis and prognosis. Microneedle (MN) integration of sampling and instant biomarker readout hold great potential in health status monitoring and point-of-care testing (POCT). The present work describes an attractive MN sensor array for minimally invasive monitoring of ISF microRNA (miRNA) and Cu2+. The MN array is made of methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (MeHA), and a further divisionally encapsulated miRNA and Cu2+ detection system, and is cross-linked through blue-light irradiation. The MN patch displays good mechanical properties that enable withstanding more than 0.4 N per needle, and exhibits a high swelling ratio of 700% that facilitates timely extraction of sufficient ISF for biomarker analysis. For proof-of-concept, it realizes detection of miRNAs and Cu2+ efficiently and quantitatively in an agarose skin and fresh porcine cadaver skin model. Given the good sampling and in situ monitoring ability, the MN array holds great promise for skin ISF-based applications.

Silver nanoclusters were synthesized and passivated by glutathione (GSH) ligand, with high aqueous stability and powerful red fluorescence and UV-vis yellow colour. Importantly, the specific recognition of the AgNCs was modulated from Hg2+ ions to Cu2+ ions upon the GSH passivation, of which the unique GSH-Cu2+ chelating reaction could conduct the fluorescence quenching of AgNCs. Strong UV-vis absorbance of GSH-passivated AgNCs could also be realized depending on the Cu2+ levels. Moreover, the Cu2+-induced loss of fluorescence and UV-vis absorbance of GSH-passivated AgNCs could be well restored by using stronger Cu2+ chelating agent. A simultaneous and reversible fluorimetric and colorimetric sensing method was thereby developed for probing Cu2+ ions in blood with high sensitivity and selectivity. Subsequently, the fluorescence-trackable imaging for live tissues and cells was demonstrated towards the analysis Cu2+ ions using GSH-passivated AgNCs as the fluorescent probes. This study indicates that the use of functional ligands like GSH could not only modulate the specific ion recognition of AgNCs, but also endow them the high aqueous stability and powerful red fluorescence towards the wide applications for ion sensing and biological imaging in the complicated media like blood.

A glass slide was first patterned with hydrophobic hexadecyltrimethoxysilane (HDS) and then microspotted with hydrophilic ZnO nanoparticles in an aminopropyltriethoxysilane (APS) matrix. The resulting HDS-ZnO-APS microarray could present the capability of suppressing the undesirable "coffee-ring" effects through its hydrophobic pattern so as to allow the fabrication of ZnO-APS testing microspots with a highly dense and uniform distribution. The lotus-like "self-cleaning" function could also be expected to effectively curb the cross contamination of multiple sample droplets. More importantly, the introduction of ZnO nanoparticles could endow the testing microspots with substrate-enhanced fluorescence leading to signal-amplification microarray fluorometry. The practical application of the developed HDS-ZnO-APS microarray was investigated by the sandwiched fluorometric immunoassays of human IgG, showing a linear detection range from 0.010 to 10.0 ng mL(-1). Such a throughput-improved fluorometric microarray could be tailored for probing multiple biomarkers in complicated media like serum or blood.

A fluorimetric Hg2+ test strip has been developed using a lotus-inspired fabrication method for suppressing the “coffee stains” toward the uniform distribution of probe materials through creating a hydrophobic drying pattern for fast solvent evaporation. The test strips were first loaded with the model probes of fluorescent gold-silver nanoclusters and then dried in vacuum on the hydrophobic pattern. On the one hand, here, the hydrophobic constraining forces from the lotus surface-like pattern could control the exterior transport of dispersed nanoclusters on strips leading to the minimized “coffee stains”. On the other hand, the vacuum-aided fast solvent evaporation could boost the interior Marangoni flow of probe materials on strips to expect the further improved probe distribution on strips. High aqueous stability and enhanced fluorescence of probes on test strips were realized by the hydrophilic treatment with amine-derivatized silicane. A test strips-based fluorimetry has thereby been developed for probing Hg2+ ions in wastewater, showing the detection performances comparable to the classic instrumental analysis ones. Such a facile and efficient fabrication route for the bio-inspired suppression of “coffee stains” on test strips may expand the scope of applications of test strips-based “point-of-care” analysis methods or detection devices in the biomedical and environmental fields.

A rapid, ultrasensitive, and high-throughput fluorimetric microarray method has been developed using hydrophobic pattern as the microarray substrate and 3-aminopropyltriethoxysilane-coupled carboxylic acid calcium (APS-CCA) as the fluorescent probes for sensing Ca2+ ions in blood. The hydrophobic pattern of the developed Ca2+ analysis microarray could largely suppress the “coffee-ring” effects to facilitate the better distribution density of testing microspots toward the high-throughput detections, and especially prevent the cross-contamination of the multiple samples between adjacent microspots. Moreover, the use of APS matrix could endow the CCA probe the enhanced environmental stability and fluorescence intensity, which is about 2.3-fold higher than that of free CCA. The interactions between APS-CCA and Ca2+ ions were systematically characterized by UV-vis and fluorescence measurements including microscopy imaging. It was demonstrated that the fluorimetric microarray could display the strong capacity of specifically sensing Ca2+ ions with the minimal interferences from blood backgrounds. Such an APS-CCA-based fluorimetric microarray can allow for the analysis of Ca2+ ions down to 0.0050 mM in blood, promising a highly sensitive and selective detection candidate for Ca2+ ions to be applied in the clinical laboratory.

DNAzyme shows great promise in designing a highly sensitive and specific sensing platform; however, the low cellular uptake efficiency, instability, and especially the insufficient cofactor supply inhibit the intracellular molecule sensor applications. Herein, we demonstrate a novel type of DNAzyme-based self-driven intracellular sensor for microRNA (miRNA) detection in living cells. The sensor consists of a metal–organic framework [zeolite imidazole framework (ZIF-8)] core loaded with a shell consisting of a rationally designed DNAzyme, where the substrate strand is modified with FAM and BHQ-1 nearby both the sides of the restriction site, respectively, while the enzyme strand consists of two separate strands with a complementary fragment to the substrate strand and the targeting miRNA, respectively. The ZIF-8 nanoparticles enable the efficient delivery of DNAzyme into the cell and protect the DNAzyme from degradation. The pH-responsive ZIF-8 degradation is accompanied with the release of the DNAzyme and Zn2+ cofactors, and the intracellular target miRNAs recognize and activate the DNAzyme driven by the Zn2+ cofactors to cleave the substrate strand, resulting in the release of the FAM-labeled shorter product strand and increased fluorescence for miRNA detection. The self-driven approach can be generally applied to various miRNAs’ detection through DNAzyme engineering.