Smart Composite Reagent Composed of Double-Stranded DNA-Templated Copper Nanoparticle and SYBR Green I for Hydrogen Peroxide Related Biosensing

On the basis of an interesting experimental phenomenon, a novel and smart composite reagent consisting of double-stranded DNA-templated copper nanoparticles (dsDNA–CuNPs) and DNA intercalator (SYBR Green I) was developed and exploited for hydrogen peroxide (H₂O₂) detection as well as oxidase-based biosensing. The study found that, within the composite reagent, the small molecule SYBR Green I was easily adsorbed on the surfaces of CuNPs, instead of intercalating into the dsDNA. So the composite reagent only exhibited the red fluorescence generated from dsDNA–CuNPs. However, when the solution of H₂O₂ was added into the composite reagent, the CuNPs were deconstructed and their fluorescence was quenched; meanwhile, the inhibition of SYBR Green I binding with dsDNA was eliminated. As a result, the mixed solution of the composite reagent with H₂O₂ exhibited green fluorescence generated from the intercalation of SYBR Green I into dsDNA. Since H₂O₂ is an important molecule and involved in various research fields, this developed composite reagent could be employed for many applications in biological analysis. As a proof-of-application demonstration, the sensitive detection of glucose was conducted. Moreover, the method was also extended to the detection of other biomolecules, such as cholesterol and horseradish peroxidase, which indicated the broad applications of the proposed sensing strategy in biomedical analysis

Multifunctional Dumbbell-Shaped DNA-Templated Selective Formation of Fluorescent Silver Nanoclusters or Copper Nanoparticles for Sensitive Detection of Biomolecules

In this work, a multifunctional template for selective formation of fluorescent silver nanoclusters (AgNCs) or copper nanoparticles (CuNPs) is put forward. This dumbbell-shaped (DS) DNA template is made up of two cytosine hairpin loops and an adenine–thymine-rich double-helical stem which is closed by the loops. The cytosine loops act as specific regions for the growth of AgNCs, and the double-helical stem serves as template for the CuNPs formation. By carefully investigating the sequence and length of DS DNA, we present the optimal design of the template. Benefiting from the smart design and facile synthesis, a simple, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection is proposed. Through the systematic comparison, it is found that the strategy based on CuNPs formation is more sensitive for ATP assay than that based on AgNCs synthesis, and the detection limitation was found to be 81 pM. What’s more, the CuNPs formation-based method is successfully applied in the detection of ATP in human serum as well as the determination of cellular ATP. In addition to small target molecule, the sensing strategy was also extended to the detection of biomacromolecule (DNA), which illustrates the generality of this biosensor

One-Pot Synthesis of DNA-CdTe:Zn²⁺ Nanocrystals Using Na₂TeO₃ as the Te source

DNA-functionalized quantum dots (QDs) are powerful tools for biosensing and bioimaging applications. Facile labeling methods with good fluorescence properties are desirable for the development of DNA-functionalized QDs. In this article, we describe a novel and simple approach that leads to the synthesis of DNA-functionalized CdTe:Zn²⁺ QDs in one step. It is the first time that DNA-functionalized QDs have been prepared using sodium tellurite as the tellurium source by a hydrothermal method. This approach will greatly reduce the synthesis time (only about 1 h) and simplify the synthesis process as well as reduce the complexity of the required experimental techniques. The as-prepared QDs exhibit high quantum yield, small size, and low toxicity. UV–vis spectra and FTIR characterization proved that the abundance of DNA on the surface of the QDs increased with the increase in the concentration of the feed DNA. Most importantly, these QDs functionalized with DNA have great potential to bind specifically to DNA, protein, and cell surface receptors

Multipedal DNA Walker Biosensors Based on Catalyzed Hairpin Assembly and Isothermal Strand-Displacement Polymerase Reaction for the Chemiluminescent Detection of Proteins

In this study, two kinds of sensitive biosensors based on a multipedal DNA walker along a three-dimensional DNA functional magnet particles track for the chemiluminescent detection of streptavidin (SA) are constructed and compared. In the presence of SA, a multipedal DNA walker was constructed by a biotin-modified catalyst as a result of the terminal protection to avoid being digested by exonuclease I. Then, through a toehold-mediated strand exchange, a “leg” of a multipedal DNA walker interacted with a toehold of a catalyzed hairpin assembly (CHA)-H1 coupled with magnetic microparticles (MMPs) and opened its hairpin structure. The newly open stem in CHA-H1 was hybridized with a toehold of biotin-labeled H2. Via the strand displacement process, H2 displaced one “leg” of a multipedal DNA walker, and the other “leg” continued to interact with the neighboring H1 to initiate the next cycle. In order to solve the high background caused by the hybridization between CHA-H1 and H2 without a CHA-catalyst, the other model was designed. The principle of the other model (isothermal strand-displacement polymerase reaction (ISDPR)-DNA walker) was similar to that of the above one. After the terminal protection of SA, a “leg” of a multipedal DNA walker was triggered to open the hairpin of the ISDPR-H1 conjugated with MMPs. Then, the biotin-modified primer hybridized with the newly exposed DNA segment, triggering the polymerization reaction with the assistance of dNTPs/polymerase. As for the extension of the primer, the “leg” of a multipedal DNA walker was displaced so that the other “leg” could trigger the proximal H1 to go onto the next cycle. Due to its lower background and stronger signal, a multipedal DNA walker based on an ISDPR had a lower limit of detection for SA. The limit of detection for SA was 6.5 pM, and for expanding the application of the method, the detections of the folate receptor and thrombin were explored. In addition, these DNA walker methods were applied in complex samples successfully

Rox-DNA Functionalized Silicon Nanodots for Ratiometric Detection of Mercury Ions in Live Cells

A ratiometric fluorescent sensor for mercury ions (Hg²⁺) has been constructed via covalent functionalization of silicon nanodot (SiND) with Hg²⁺-specific 6-carboxy-X-rhodamine (Rox)-tagged DNA. For the Rox-DNA functionalized SiND, the red fluorescence of Rox can be quenched by the blue-emitting SiND in the presence of Hg²⁺ due to structural change in DNA, which serves as the response signal. Meawhile, the fluorescence of SiND is insensitive to Hg²⁺ and acts as the reference signal. The wavelength difference in the optimal emission peak is as large as 190 nm between SiND (422 nm) and Rox (612 nm), which can efficaciously exclude the interference of the two emission peaks, and facilitates dual-color visualization of Hg²⁺ ions. The biofunctionalization of SiND improves the acid–base stability of SiND significantly, which is favorable for its application in the intracellular environment. Accordingly, a sensitive, simple, precise and rapid method for tracing Hg²⁺ was proposed. The limit of detection and precision of this method for Hg²⁺ was 9.2 nM and 8.8% (50 nM, <i>n</i> = 7), respectively. The increase of Hg²⁺ concentration in the range of 10–1500 nM was in accordance with linearly increase of the <i>I</i>₄₂₂/<i>I</i>₆₁₂ ratio. As for practical application, the recoveries in spiked human urine and serum samples were in the range of 81–107%. Moreover, this fluorescent nanosensor was utilized to the ratiometric detection of Hg²⁺ in HeLa cells

Periodic Fluorescent Silver Clusters Assembled by Rolling Circle Amplification and Their Sensor Application

A simple method for preparing DNA-stabilized Ag nanoclusters (NCs) nanowires is presented. To fabricate the Ag NCs nanowires, we use just two unmodified component strands and a long enzymatically produced scaffold. These nanowires form at room temperature and have periodic sequence units that are available for fluorescence Ag NCs assembled which formed three-way junction (TWJ) structure. These Ag NCs nanowires can be clearly visualized by confocal microscopy. Furthermore, due to the high efficiency of rolling circle amplification reaction in signal amplification, the nanowires exhibit high sensitivity for the specific DNA detection with a wide linear range from 6 to 300 pM and a low detection limit of 0.84 pM, which shows good performance in the complex serum samples. Therefore, these Ag NCs nanowires might have great potential in clinical and imaging applications in the future

Peptide-Capped Gold Nanoparticle for Colorimetric Immunoassay of Conjugated Abscisic Acid

The pentapeptide Cys-Ala-Leu-Asn-Asn (CALNN) has been proved to be a powerful tool to stabilize the AuNPs. These CALNN-capped AuNPs have been used to develop various bioanalysis platforms. In this paper, the CALNN-capped AuNPs are proved to be a robust tool for aggregation-based colorimetric immunoassays as well. A colorimetric immunoassay strategy based upon the antibody-induced assembly of functionalized AuNPs for Abscisic Acid glucose ester (ABA-GE) determination has been developed. The ABA-functionalized AuNPs aggregate in the presence of specific antibody, accompanied by a color change of the solution. The color change is competitively inhibited by ABA-GE. The interparticle distance in aggregates is small due to the thin peptide layer on the AuNPs surface, and it is determined by the “Y” shape antibody linker as well. As a result of that, an obvious color change in the immunoassays is observed. Under the optimized conditions, a linear response range from 5 nM to 10 μM for ABA-GE determination is obtained, and the limit of detection (LOD) is evaluated to be 2.2 nM. This method is simple, homogeneous, and has potential for visual detection of ABA-GE

One-Step Synthesis of Rox-DNA Functionalized CdZnTeS Quantum Dots for the Visual Detection of Hydrogen Peroxide and Blood Glucose

As the blood glucose concentration is an important clinical parameter of diabetes, the rapid and effective detection of blood glucose is very significant for monitoring and managing diabetes. Here, a facile method to prepare Rox-DNA functionalized CdZnTeS quantum dots (QDs) was developed. The Rox-DNA functionalized CdZnTeS QDs were prepared by a one-pot hydrothermal method through phosphorothioate DNA bound to QDs, which were employed as a ratiometric fluorescent probe for the rapid and sensitive detection of H₂O₂ and glucose. Compared with the traditional multistep construction of ratiometric fluorescent probes, this presented approach is simpler and more effective without chemical modification and complicated separation. The CdZnTeS QDs with green fluorescence is specifically sensitive to H₂O₂, while the red fluorescence of Rox is invariable. H₂O₂ is the product from the oxidation of glucose catalyzed by glucose oxidase (GOx). Therefore, a facile method to detect H₂O₂ and glucose with a detection limit of 0.075 μM for H₂O₂ and 0.042 μM for glucose was developed. In addition, this proposed probe has been employed for the detection of glucose in human serum with a satisfactory result. Moreover, this probe has been used for visual detection, and the health and diabetics can be distinguished by the naked eye. Meanwhile, this nanoprobe is also generalizable and can be extended to the detection of many other H₂O₂-mediated analytes

Controlled Assembly of Gold Nanoparticles through Antibody Recognition: Study and Utilizing the Effect of Particle Size on Interparticle Distance

An assembly of gold nanoparticle through the recognition of unmodified antibody was developed. The use of peptide (Cys-Ala-Leu-Asn-Asn) as ligands to stabilize and functionalize gold nanoparticles provides technical and operational convenience. These peptide-capped particles in different sizes are recognized by antibody and assembly to form dimers and expanded hybrid material by controlling the conditions. The interparticle spacing of these assemblies was well studied with small-angle X-ray scattering measurements, and it was found that the interparticle spacing is inversely dependent on the particle size. This relationship of interparticle spacing and particle size is closely related to the structure of antibody linker. Therefore, analyzing the interparticle spacing of assemblies can reveal the equilibrium configuration of IgG. Based on the investigation, the Fab–Fab angle of IgG is obtained to be ≈102° and the Fab arms are ≈7.8 nm. These results provide new experimental data on the structure of flexible IgG

Simultaneous Determination of Human Enterovirus 71 and Coxsackievirus B3 by Dual-Color Quantum Dots and Homogeneous Immunoassay

Human Enterovirus 71 (EV71) and Coxsackievirus B3 (CVB3) have high risks for morbidity and mortality. A virus quantitation immunoassay has been proposed by employing two colored quantum dots (QDs), antibodies of the virus, and graphene oxide (GO). The QDs are streptavidin-conjugated quantum dots (SA-QDs), and the antibodies are biotinylated antibodies. Biotinylated EV71 antibody (Ab1) was associated with 525 nm green colored SA-QDs via biotin-streptavidin interaction forming QDs-Ab1, whereas biotinylated CVB3 antibody (Ab2) was associated with 605 nm red colored SA-QDs via biotin–streptavidin interaction forming QDs-Ab2. GO was an excellent quencher to the fluorescence of both QDs-Ab1 and QDs-Ab2. The targets of EV71 and CVB3 can break up the complex of QDs-Ab and GO, recovering the fluorescence of QDs-Ab1 and QDs-Ab2, respectively. Using these two different colored QDs-Ab fluorescence recovery intensities upon the addition of targets EV71 and CVB3, the two enteroviruses can be simultaneously quantitatively determined with a single excitation light. The detection limits of EV71 and CVB3 are 0.42 and 0.39 ng mL^–1 based on 3 times signal-to-noise ratio, respectively. More importantly, this strategy can be further used as a universal method for any protein or virus determination by changing the conjugated antibodies in disease early diagnosis, which can provide a fast and promising clinical approach for virus differentiation and determination. In a word, a simple, fast, sensitive, and highly selective assay for EV71 and CVB3 has been developed. It could be applied in clinical sample analysis with a satisfactory result. It was notable that the sensor could not only achieve rapid and precise quantitative determination of protein/virus by fluorescent intensity but also could be applied in semiquantitative protein/virus determination by digital visualization