Label-free and portable detection of HIV-DNA by a handheld luminometer.

BACKGROUND: The human immunodeficiency virus (HIV) remains a major worldwide health problem. Nowadays, many methods have been developed for quantitative detecting human immunodeficiency virus DNA (HIV-DNA), such as fluorescence and colorimetry. However, these methods still have the disadvantages of being expensive and requiring professional technicians. Early diagnosis of pathogens is increasingly dependent on portable instruments and simple point-of-care testing (POCT). Therefore, it is meaningful and necessary to develop portable and cheap methods for detecting disease markers. RESULTS: In this work, a label-free chemiluminescence (CL) method was developed for detecting HIV-DNA via a handheld luminometer. To achieve label-free target detection, the CL catalyst, G-triplex-hemin DNAzyme (G3-hemin DNAzyme), was in-situ assembled in the presence of HIV-DNA. For improving sensitivity, HIV-DNA induced the cyclic strand displacement reaction (SDR), which can form three G3-hemin DNAzymes in one cycle. So, the chemiluminescence reaction between luminol and H2O2 was highly effectively catalyzed, and the CL intensity was linearly related with the concentration of HIV-DNA in the range of 0.05-10 nM with a detection limit of 29.0 pM. Due to the high specificity of hairpin DNA, single-base mismatch can be discriminated, which ensured the specific detection of HIV-DNA. SIGNIFICANCE: In-situ formation of G3-hemin DNAzyme led to label-free and selective detection without complex synthesis and functionalization. Therefore, it offers a cheap, selective, sensitive and portable method for detecting disease-related genes, which is promising for POCT of clinical diagnosis in resource-limited settings.

Multifunctional Manganese-Nucleotide Laccase-Mimicking Nanozyme for Degradation of Organic Pollutants and Visual Assay of Epinephrine via Smartphone.

As a natural green catalyst, laccase has extensive application in the fields of environmental monitoring and pollutant degradation. However, susceptibility to environmental influences and poor reusability seriously hinder its application. To address these concerns, for the first time, manganese ion replaced copper ion as the active center to coordinate with guanosine monophosphate (GMP) for synthesizing mimic laccase with high catalytic activity. Compared with natural laccase, the laccase-like nanozyme (Mn-GMPNS) demonstrated superior thermal stability, acid-base resistance, salt tolerance, reusability, and substrate universality. Benefiting from the high catalytic activity of Mn-GMPNS, epinephrine, a significant neurotransmitter and hormone associated with numerous diseases, was visually detected within 10 min and a portable assay by smartphone. More encouragingly, Mn-GMPNS can efficiently degrade dye pollutants, achieving a decolorization rate over 70% within 30 min. Thus, the coordination between manganese ion and nucleotide demonstrated the potential in rational design of nanozymes with high catalytic activity, low cost, good stability, and good biocompatibility.

Entropy-driven catalysis-based lateral flow assay for sensitive detection of Alzheimer 's-associated MicroRNA.

Alzheimer's disease (AD) is a degenerative disease of the brain worldwide. Currently, there is no effective cure. But accurate and early diagnosis of AD is critical to the development of patient care and future treatments. MiRNA-16 has been considered as an effective diagnostic biomarker for AD because of its regulatory effect on key proteins of AD. Herein, a colorimetric lateral flow assay (LFA) was developed for sensitive detection of miRNA-16 based on entropy-driven catalysis (EDC) amplification strategy. MiRNA-16 triggered EDC and released more linker DNAs (LDNA) of sandwich structure. Thus, AuNPs were enriched at the T-line to enhance the colorimetric signal and improve the sensitivity of visual assay. It showed good specificity and sensitivity for detecting miRNA-16 with a detection limit of 1.01 pM. The practical detection of miRNA-16 in human serum obtained satisfactory result. Significantly, EDC achieved signal amplification in homogeneous solution without enzyme and DNA labeling, leading to a cheap and easy detection of miRNA-16. Therefore, it provided a portable and rapid assay for AD-related nucleic acid, which holds a potential for point-of-care testing (POCT) of AD.

Self-assembly of DNA-gold nanoaggregate for visual detection of thymidine kinase 1 (TK1) mRNA via lateral flow assay.

Nucleic acid lateral flow assay (NALFA) with gold nanoparticles (AuNPs) as colorimetric probes have been extensively adopted for point-of-care testing (POCT). However, the sensitivity of NALFA still needs to be improved. Herein, DNA-gold nanoaggregate (DNA-AuNA) was assembled as a signal amplification probe of NALFA for sensitive detection of tumor marker TK1 mRNA. Four functional oligonucleotides with complementary pairs were assembled to form DNA-AuNA that coupled more AuNPs to improve sensitivity. Thus, the limit of detection (LOD) was 0.36 pM, which is lower than that of conventional AuNPs-based NALFA. Moreover, the bioassay showed good reproducibility, stability, and specificity for detecting TK1 mRNA. The detection of TK1 mRNA in human serum was also satisfactory. Therefore, DNA-AuNA-based NALFA provides a sensitive method for portable detection of TK1 mRNA.

Multifunctional DNA nanoprobe for tumor-targeted synergistic therapy by integrating chemodynamic therapy with gene silencing.

Due to the high complexity, diversity and heterogeneity of tumor occurrence and development, multi-mode synergistic therapy is more effective than single treatment modes to improve the antitumor efficacy. Also, multifunctional probes are crucial to realize synergistic therapy. Herein, a multifunctional DNA tetrahedron nanoprobe was ingeniously designed to simultaneously achieve chemodynamic therapy (CDT) and gene silencing for synergistic antitumor. The multifunctional DNA tetrahedron nanoprobe, DNA tetrahedron-silver nanocluster-antagomir-21 (D-sgc8-DTNS-AgNCs-Anta-21), integrated a CDT reagent (DNA-AgNCs) and miRNA-21 inhibitor (Anta-21) with a specific recognition probe (aptamer). After targeted entry in cancer cells, D-sgc8-DTNS-AgNCs-Anta-21 silenced endogenous miRNA-21 by Anta-21 and produced highly toxic ˙OH by reacting with H2O2, which induced apoptosis in the tumor cells. The targeted recognition of aptamers led to the concentration-dependent death of HeLa cells. On the contrary, the cell survival rate of normal cells was basically unaffected with an increase in the concentration of D-sgc8-DTNS-AgNCs-Anta-21. Therefore, the diverse functions, biocompatibility and programmability of DNA provide a useful and easy way to assemble multifunctional probes for synergistic therapy.

A biomineralized bi-functional hybrid nanoflower to effectively combat bacteria via a glucose-powered cascade catalytic reaction.

The bacterial resistance due to the abuse of conventional antibiotics is regarded as a major problem for bacterial-induced infections and chronic wound healing. There is an urgent need to explore alternative antimicrobial strategies and functional materials with excellent antibacterial efficacy. Herein, guanosine monophosphate (GMP) and glucose oxidase (GOD) were coordinated with copper ions to obtain a bi-functional hybrid nanoflower (Cu-GMP/GODNF) as a cascade catalyst for promoting antibacterial efficacy. Besides the efficient conversion of glucose to hydrogen peroxide, the produced gluconic acid by loading GOD can supply a compatible catalytic environment to substantially improve the peroxidase activity for generating more toxic reactive oxygen species (ROS). So, the glucose-powered cascade catalytic reaction effectively killed bacteria. Moreover, H2O2 self-supplied by glucose can reduce harmful side effects of exogenous H2O2. Meanwhile, the adhesion between the Cu-GMP/GODNF and the bacterial membrane can enhance the antibacterial efficacy. Therefore, the achieved bi-functional hybrid nanoflower exhibited high efficiency and biocompatibility for killing bacteria in diabetes-related infections.

Dual-Mode Logic Gate for Intelligent and Portable Detection of MicroRNA Based on Gas Pressure and Lateral Flow Assay.

Molecular logic gate provides an intelligent option for simultaneous detection of biomarkers. Herein, a dual-mode DNA logic gate was proposed to portably and intelligently detect multiple microRNAs (miRNAs) by gas pressure biosensing and lateral flow assay (LFA). A platinum-coated gold nanoparticle (Au@PtNP) with catalase-like activity was used as a signal reporter to achieve a dual-signal readout. MiRNAs as the input initiated the cyclic strand displacement reaction (SDR) to enrich a large amount of Au@PtNPs. Thus, miRNA can be visually detected by a lateral flow strip (LFS) using the grayish-brown color of Au@PtNPs as output 1. Furthermore, Au@PtNP-catalyzed decomposition of H2O2 resulted in gas pressure as output 2, which was measured by a digital and handheld gas pressure meter. As a consequence, microRNA 21 (miR-21) was sensitively and reliably detected with the limit of detection (LOD) of 7.2 pM. The selectivity and real sample analysis were both satisfactory. Significantly, two-input and three-input AND logic gates were successfully developed to realize multiple detection of two miRNAs and three miRNAs, which provide a promising way for intelligent multi-input analysis. Predictably, with the advantages of portability, simplicity, and affordability, the dual-mode logic gate based on gas pressure biosensing and LFA offers a new perspective on the field of intelligent and portable biosensing and bioanalysis.

Logic-Gates of Gas Pressure for Portable, Intelligent and Multiple Analysis of Metal Ions.

DNA logic gates have shown outstanding magic in intelligent biology applications, but it remains challenging to construct a portable, affordable and convenient DNA logic gate. Herein, logic gates of gas pressure were innovatively developed for multiplex analysis of metal ions. Hg2+ and Ag+ were input to interact specifically with the respective mismatched base pairs, which activated DNA extension reaction by polymerase and led to the enrichment of platinum nanoparticles for catalyzing the decomposition of peroxide hydrogen. Thus, the gas pressure obtained from a sealed well was used as output for detecting or identifying metal ions. Hg2+ and Ag+ were sensitively and selectively detected, and the assay of the real samples was also satisfactory. Based on this, DNA logic gates, including YES, NOT, AND, OR, NAND, NOR, INHIBIT, and XOR were successfully established using a portable and hand-held gas pressure meter as detector. So, the interactions between DNA and metal ions were intelligently transferred into the output of gas pressure, which made metal ions to be detected portably and identified intelligently. Given the remarkable merits of simplicity, logic operation, and portable output, the metal ion-driven DNA logic gate of gas pressure provides a promising way for intelligent and portable biosensing.

Automated study on kinetics and biosensing of glow-type luminescence reaction via digital microfluidics-chemiluminescence.

Automated manipulation of discrete droplets by digital microfluidics (DMF) combined with chemiluminescence (CL) is promising to achieve automated and sensitive biosensing and bioanalysis. Herein, a DMF-CL device was built to automatically study CL kinetics and biosensing of a glow-type CL reaction. Copper-cysteine nanoparticles (Cu/CysNP) were synthesized as a new CL catalyst to extend the CL reaction of luminol-H2O2 to more than 10 min. The automated manipulation of droplets reduced reagent costs and manual errors, leading to real-time, automated, and reliable study of CL kinetics. The CL kinetics curve collected by the DMF-CL integration device is in accordance with that of a commercial CL analyser. The long-lasting luminescence ensured automated, sensitive, and reliable detection of H2O2 as a direct or indirect analyte of the cascade catalytic reaction. Moreover, an innovative asymmetrical splitting method is proposed to quickly and precisely generate daughter droplets to ensure uniformity of the droplets and good repeatability of the DMF-CL measurements. Therefore, the DMF-CL analysis holds great potential for achieving online and automatic analysis and biosensing.

Peroxidase-Mimicking DNAzymes as Receptors for Label-Free Discriminating Heavy Metal Ions by Chemiluminescence Sensor Arrays.

Receptors are crucial to the analytical performance of sensor arrays. Different from the previous receptors in sensor arrays, herein, peroxidase-mimicking DNAzymes were innovatively used as receptors to develop a label-free chemiluminescence sensor array for discriminating various heavy metal ions in complex samples. The peroxidase-mimicking DNAzymes are composed of functional oligonucleotides and hemin, including G-triplex-hemin DNAzyme (G3-DNAzyme), G-quadruplex-hemin DNAzyme (G4-DNAzyme), and the dimer of G-quadruplex-hemin DNAzyme (dG4-DNAzyme). Circular dichroism (CD) spectroscopy demonstrated that different metal ions diversely affect the conformation of G-quadruplex and G-triplex, resulting in a change in the activity of peroxidase-mimicking DNAzyme. Thus, the unique fingerprints formed to easily discriminate seven kinds of heavy metal ions by principal component analysis (PCA) within 20 min. The discrimination of unknown metal ions in tap water further confirmed its ability for discriminating multiple heavy metal ions. Moreover, it will not bring water pollution due to the good biocompatibility of DNA. Therefore, it not only merely offers a label-free, rapid, environment-friendly, and cheap (1.49 $) sensor assay for discriminating metal ions but also comes up with an innovative way for developing sensor arrays.

Acid-accelerated hydrolysis of NaBH4: a gas-generation reaction for diverse gas pressure biosensing.

Gas pressure biosensing is a promising portable analysis method. The gas-generation reaction is crucial to its sensitivity, speed, repeatability, and usability. However, very few gas-generation reactions are available for sensitive, safe, and diverse biosensing. Herein, acid-accelerated hydrolysis of sodium borohydride (NaBH4) was explored for the first time to achieve portable and diverse gas pressure biosensing. The slow hydrolysis and hydrogen generation of NaBH4 in alkaline medium is accelerated with increasing acidity, which increased the gas pressure in a small and sealed tube within 10 min. Thus, a label-free bioassay is easily and specifically achieved once analytes can in-situ generate acid to accelerate the hydrolysis rate of NaBH4, such as glucose, acetylcholine (ACh), adenosine triphosphate (ATP) and others. More importantly, analytes without acid generation could be quantitatively and selectively detected by combining target recognition with acid-generated biochemical reactions for enzyme-linked gas pressure biosensing. Inspired by this, aflatoxin B1 (AFB1)-aptamer interaction-triggered strand displacement reaction was combined with glucose oxidation by glucose oxidase (GOD) to detect AFB1 as low as 7.1 pM. Therefore, acid-accelerated hydrolysis of NaBH4 is powerful for developing portable, cheap, and diverse gas pressure biosensing. It opens up a new way for cheap, universal, and portable biosensing.

Portable and Label-Free Sensor Array for Discriminating Multiple Analytes via a Handheld Gas Pressure Meter.

Cross-reactive sensor arrays are useful for discriminating multiple analytes in a complex sample. Herein, a portable and label-free gas pressure sensor array was proposed for multiplex analysis via a handheld gas pressure meter. It is based on the interaction diversity of analytes with catalase-like nanomaterials, including Pt nanoparticles (PtNP), Co3O4 nanosheets (Co3O4NS), and Pt-Co alloy nanosheets (PtCoNS), respectively. Thus, the diverse influence of analytes on the catalase-like activity could be output as the difference in the gas pressure. By using principal component analysis, eight proteins were well distinguished by the gas pressure sensor array at the 10 nM level within 12 min. Moreover, different concentrations of proteins and mixtures of proteins could likewise be discriminated. More importantly, the effective discrimination of proteins in human serum and discrimination of five kinds of cells further confirmed the potential of the gas pressure sensor array. Therefore, it provides a portable, cheap, sensitive, and label-free gas pressure sensor array, which is totally different from the reported sensor arrays and holds great potential for portable and cheap discrimination of multiple analytes.

Nanotrains of DNA Copper Nanoclusters That Triggered a Cascade Fenton-Like Reaction and Glutathione Depletion to Doubly Enhance Chemodynamic Therapy.

Many current chemodynamic therapy (CDT) strategies suffer from either low therapeutic efficiency or the deficiency of poor targeting. The low therapeutic efficiency is mainly ascribed to the intracellular antioxidant system and the inefficient Fenton reaction in the weakly acidic tumor microenvironment (TME). Herein, by exploitation of the diverse function and programmability of functional nucleic acid, aptamer-tethered nanotrains of DNA copper nanoclusters (aptNTDNA-CuNCs) were assembled to simultaneously achieve targeted recognition, loading, and delivery of CDT reagents into tumor cells without an external carrier. The intracellular hydrogen peroxide (H2O2) oxidized nanotrains of DNA-CuNCs to produce a lot of Cu2+ and Cu+ ions, which can generate reactive oxygen species (ROS) in the weakly acidic TME based on the pH-independent Fenton-like reaction of Cu+/H2O2. Meanwhile, the redox reaction between intracellular glutathione (GSH) and Cu2+ depleted GSH and generated Cu+ ions, which weakened the antioxidant ability of cancer cells and further enhanced the Fenton-like reaction of Cu+/H2O2, respectively. Thus, the cascade Fenton-like reaction and GSH depletion doubly improved the efficacy of CDT. The in vivo and in vitro study solidly confirmed that aptNTDNA-CuNCs have excellent antitumor efficacy and no cytotoxicity to healthy cells. Therefore, aptNTDNA-CuNCs can act as CDT reagents to achieve highly efficient, biocompatible, and targeted CDT.

Enzyme-Free Colorimetric Immunoassay for Protein Biomarker Enabled by Loading and Disassembly Behaviors of Polydopamine Nanoparticles.

In this work, a mussel-inspired polydopamine (PDA) nanoparticle with biocompatible and reactive surface characteristics was desirably incorporated with high loading capacity and pH-induced disassembly-releasing behavior toward Fe(III) ions to develop an enzyme-free colorimetric immunoassay. The catechol functional network of PDA can act as a scaffold to coordinate with large amounts of Fe(III) ions to form the PDA-Fe(III) NPs whose coordination state can be tailored by changing the pH values. In detail, PDA-Fe(III) NPs can be maintained in a disassembled tri-coordinate state under alkaline conditions while the highly loaded Fe(III) ions can be easily released under acid conditions to react with ferrocyanide for the in situ generation of Prussian blue (PB) NPs. The detection sensitivity of prostate-specific antigen (PSA) was significantly improved, owing to the high peroxidase-like activity of PB NPs that triggered excellent catalytic effect by the colorimetric reaction. In addition, favorable linearity was found in the range of 0.0005-20 ng mL-1 with a low detection limit of 0.84 pg mL-1 (S/N = 3). Furthermore, excellent selectivity and reproducibility were exhibited by the developed enzyme-free colorimetric immunoassay. It is believed that this proposed PDA-Fe(III) NP-based enzyme-free colorimetric system will offer a facile and reliable tool for the sensitive detection of PSA and other cancer biomarkers in human serum.

Ratiometric fluorescent immunoassay for the cardiac troponin-I using carbon dots and palladium-iridium nanocubes with peroxidase-mimicking activity.

A nanozyme-linked immunosorbent assay is described for cardiac troponin I which is a biomarker for myocardial infarction. The method is based on the use of Pd-Ir nanocubes with excellent peroxidase-like activity. The nanocubes catalyze the oxidization of nonfluorescent o-phenylenediamine (OPD) by H2O2 to form a yellow fluorescent product (oxOPD) with excitation/emission maxima at 400/570 nm. Carbon dots are added as a reference fluorophore. Under the same excitation wavelength, they display blue fluorescence (450 nm). The ELISA uses the Pd-Ir nanocubes as a label for the secondary antibody and OPD as substrate. The ratio of fluorescence intensities at 570 and 450 nm increases in the 1 pg·mL-1 to 1 ng·mL-1 cardiac troponin I concentration range, and the detection limit is 0.31 pg·mL-1. The method was applied to analyze spiked serum samples, and the results compared well with those obtained by a commercial chemiluminescence assay. Graphical abstract Schematic presentation of the ratiometric fluorescence immunoassay for cardiac troponin-I. Pd-Ir nanocubes were employed to fabricate nanozyme-based signal labels for its excellent peroxidase-mimicking activity.

Bioinspired synthesis of organic-inorganic hybrid nanoflowers for robust enzyme-free electrochemical immunoassay.

Protein-inorganic nanoflowers have been extensively used for sensing and biosensing applications by virtue of the signal enhancement of protein component---enzyme. Can the inorganic component of protein-inorganic nanoflowers be employed to amplify signal transducing for enzyme-free detection? In this work, a new kind of BSA-antibodies-copper phosphate hybrid nanoflowers (BSA-Ab2-Cu3(PO4)2) has been prepared by one-pot biomimetic mineralization process as signal enhancer for enzyme-free electrochemical immunoassay. C-reactive protein (CRP) has been chosen as a model biomarker. To the best of our knowledge, it is the first trial using the inorganic component---phosphate ions of BSA-Ab2-Cu3(PO4)2 for transducing electrochemical readout, which features the following advantages: (1) the three-dimensional hierarchical porous nanoflower morphology with a high specific surface area could load more antibodies, and BSA for blocking non-specific sites, greatly increasing the sensitivity of the fabricated immunosensors, (2) the Cu3(PO4)2 hybrid nanoflowers can supply a huge amount of phosphate anions to react with molybdate yielding molybdophosphate precipitates and generating redox currents for more robust enzyme-free electrochemical signal readout. The fabricated immunosensor has exhibited good detection performance with a linear range of 5 pg/mL-1 ng/mL and a limit of detection of 1.26 pg/mL. Moreover, our method has presented good feasibility for clinical sample analysis.