Xiao-Jing Xing

Graphene oxide based fluorescent aptasensor for adenosine deaminase detection using adenosine as the substrate.

We present a novel fluorescent aptasensor for simple and accurate detection of adenosine deaminase (ADA) activity and inhibition on the basis of graphene oxide (GO) using adenosine (AD) as the substrate. This aptasensor consists of a dye-labeled single-stranded AD specific aptamer, GO and AD. The fluorescence intensity of the dye-labeled AD specific aptamer is quenched very efficiently by GO as a result of strong π-π stacking interaction and excellent electronic transference of GO. In the presence of AD, the fluorescence of the GO-based probe is recovered since the competitive binding of AD and GO with the dye-labeled aptamer prevents the adsorption of dye-labeled aptamer on GO. When ADA was introduced to this GO-based probe solution, the fluorescence of the probe was quenched owing to ADA can convert AD into inosine which has no affinity to the dye-labeled aptamer, thus allowing quantitative investigation of ADA activity. The as-proposed sensor is highly selective and sensitive for the assay of ADA activity with a detection limit of 0.0129U/mL in clean buffer, which is more than one order of magnitude lower than the previous reports. Meanwhile, a good linear relationship with the correlation coefficient of R=0.9922 was obtained by testing 5% human serum containing a series of concentrations of ADA. Additionally, the inhibition effect of erythro-9-(2-hydroxy-3-nonyl) adenine on ADA activity was investigated in this design. The GO-based fluorescence aptasensor not only provides a simple, cost-effective and sensitive platform for the detection of ADA and its inhibitor but also shows great potential in the diagnosis of ADA-relevant diseases and drug development.

Graphene Oxide-Based Fluorescent Biosensor for Protein Detection via Terminal Protection of Small-Molecule-Linked DNA

A fluorescence method for protein detection is developed based on terminal protection of small-molecule-linked DNA by target protein and a graphene oxide-assisted DNA assay strategy. This design results in fluorescence-enhanced detection that is sensitive and selective for the target protein.

DNA-stabilized silver nanoclusters and carbon nanoparticles oxide: A sensitive platform for label-free fluorescence turn-on detection of HIV-DNA sequences.

Based on the remarkable difference between the interactions of carbon nanoparticles (CNPs) oxide with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and the fact that fluorescence of DNA-stabilized silver nanoclusters (AgNCs) can be quenched by CNPs oxide, DNA-functionalized AgNCs were applied as label-free fluorescence probes and a novel fluorescence resonance energy transfer (FRET) sensor was successfully constructed for the detection of human immunodeficiency virus (HIV) DNA sequences. CNPs oxide were prepared with the oxidation of candle soot, hence it is simple, time-saving and low-cost. The strategy of dual AgNCs probes was applied to improve the detection sensitivity by using dual- probe capturing the same target DNA in a sandwich mode and as the fluorescence donor, and using CNPs oxide as the acceptor. In the presence of target DNA, a dsDNA hybrid forms, leading to the desorption of the ssDNA-AgNCs probes from CNPs oxide, and the recovering of fluorescence of the AgNCs in a HIV-DNA concentration-dependent manner. The results show that HIV-DNA can be detected in the range of 1-50nM with a detection limit of 0.40nM in aqueous buffer. The method is simple, rapid and sensitive with no need of labeled fluorescent probes, and moreover, the design of fluorescent dual-probe makes full use of the excellent fluorescence property of AgNCs and further improves the detection sensitivity.

An ultra-high sensitive platform for fluorescence detection of micrococcal nuclease based on grapheneoxide

Micrococcal nuclease (MNase) is the extracellular nuclease of Staphylococcus aureus (S. aureus). It preferentially digests single-stranded nucleic acids. The existence of MNase can be the standard to identify S. aureus and the content of MNase can be used to evaluate the pathogenicity of S. aureus. Herein, an ultra-high sensitive and selective fluorescent sensing platform for MNase is developed based on MNase-induced DNA strand scission and the difference in affinity of graphene oxide (GO) for single-stranded DNA containing different numbers of bases in length. In the absence of MNase, the adsorption of the dye-labeled ssDNA on GO makes the dyes close proximity to GO surface resulting in high efficiency quenching of fluorescence of the dyes. Conversely, and very importantly, in the presence of MNase, it cleaves the dye-labeled ssDNA into small fragments. The introduction of GO into the sensing solution results in weak quenching of the fluorescence of the dyes due to the weak affinity of the short dye-labeled oligonuleotide fragment to GO, and the fluorescence intensity gradually increases with increasing concentration of MNase. MNase can be detected in a range of 8×10⁻⁵ to 1.6×10⁻³ unit/mL with a detection limit of 2.7×10⁻⁵ unit/mL and good selectivity. The detection limit is of two orders of magnitude lower than those reported fluorescence MNase assays. Moreover, when the GO-based biosensor is used in S. aureus sample assays, preeminent fluorescence signals are obtained, thus the platform of the GO-based biosensor can be used to detect MNase in real-world samples.

A fluorescent aptasensor using double-stranded DNA/graphene oxide as the indicator probe

We developed a fluorescent aptasensor based on the making use of double-stranded DNA (dsDNA)/graphene oxide (GO) as the signal probe and the activities of exonuclease I (Exo I). This method takes advantage of the stronger affinity of the aptamer to its target rather than to its complementary sequence (competitor), and the different interaction intensity of dsDNA, mononucleotides with GO. Specifically, in the absence of target, the competitor hybridizes with the aptamer, preventing the digestion of the competitor by Exo I, and thus the formed dsDNA is adsorbed on GO surface, allowing fluorescence quenching. When the target is introduced, the aptamer preferentially binds with its target. Thereby, the corresponding nuclease reaction takes place, and slight fluorescence change is obtained after the introduction of GO due to the weak affinity of the generated mononucleotides to GO. Adenosine (AD) was chosen as a model system and tested in detail. Under the optimized conditions, smaller dissociation constant (Kd, 311.0 µM) and lower detection limit (LOD, 3.1 µM) were obtained in contrast with traditional dye-labeled aptamer/GO based platform (Kd=688.8 µM, LOD=21.2 µM). Satisfying results were still obtained in the evaluation of the specificity and the detection of AD in human serum, making it a promising tool for the diagnosis of AD-relevant diseases. Moreover, we demonstrated the effect of the competitor on the LOD, and the results reveal that the sensitivity could be enhanced by using the rational competitor. The present design not only constructs a label-free aptamer based platform but also extends the application of dsDNA/GO complex in biochemical and biomedical studies.

Graphene oxide and metal-mediated base pairs based "molecular beacon" integrating with exonuclease I for fluorescence turn-on detection of biothiols.

A novel fluorescence turn-on strategy, based on the resistance of metal-mediated molecular-beacons (MBs) toward nuclease digestion and the remarkable difference in the affinity of graphene oxide (GO) with MBs and the mononucleotides, is designed for the biothiols assay. Specifically, the metal-mediated base pairs facilitate the dye labeled MBs to fold into a hairpin structure preventing the digestion by exonuclease I, and thus allow the fluorescence quenching. The competition binding by biothiols removes metal ions from the base pairs, causing the nuclease reaction, and less decrease in the fluorescence is obtained after incubating with GO due to the weak affinity of the product-mononucleotides to GO. Hg(2+)-mediated MBs were firstly designed for the biothiols detection, and glutathione (GSH) was applied as the model target. Under the optimal conditions, the approach exhibits high sensitivity to GSH with a detection limit of 1.53 nM. Ag(+)-mediated MBs based sensor was also constructed to demonstrate its versatility, and cysteine was studied as the model target. The satisfactory results in the determination of biothiols in serum demonstrate that the method possesses great potential for detecting thiols in biological fluids. This new approach is expected to promote the exploitation of metal-mediated base pairs-based biosensors in biochemical and biomedical studies.

An exonuclease III-aided “turn-on” fluorescence assay for mercury ions based on graphene oxide and metal-mediated “molecular beacon”

A novel fluorescence “turn-on” strategy, which is based on the formation of Hg2+-mediated molecular-beacons (MBs), the preferable cleavage capacity of exonuclease III to double-stranded DNA compared with single-stranded one, and the remarkable difference in the binding ability of graphene oxide (GO) with single-stranded DNA and the mononucleotides, is designed for Hg2+ assay. The Hg2+-mediated base pairs facilitate the dye labeled MBs to fold into a hairpin structure, which is more likely to be digested by exonuclease III, and an obvious increase in the fluorescence intensity is observed after incubating with GO due to the weak affinity of the product-mononucleotides to GO. A fluorescent “turn-on” method based on graphene oxide and exonuclease III was designed for Hg2+ assay. The introduction of GO greatly increases the signal-to-background ratio, and the sensitivity is significantly improved due to the amplified capability of exonuclease III. Under the optimal conditions, Hg2+ is specifically and sensitively detected with a detection limit of 0.83 nM. Compared with the reported Hg2+ assay methods, the proposed strategy is simple, cost effective and selective, which might provide a new platform for developing a sensitive Hg2+ biosensor. Mercury level in the blood is an important indicator of mercury poisoning in clinical study. To testify the possibility of the use of this method for the assay of Hg2+ in real samples, detection of Hg2+ in 1% human serum was investigated and satisfactory results were obtained, which suggests that this method has great potential for bioanalysis.

Graphene oxide enhanced specificity at aptamer and its application to multiplexed enzymatic activity sensing

We explore the effect of sufficient GO on the property of a dye labeled adenosine 5′-triphosphate (ATP) aptamer (P) which shows similar affinity and specificity for ATP and its analogues including adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), and adenosine (AD). It is found that ATP and its analogues give rise to fluorescence recovery of GO-quenched P to a different extent (in the order of ATP > AD > ADP > AMP), and the difference becomes larger when increasing the concentration of GO in a certain range, implying an improvement of specificity of the ATP aptamer. Based on this finding, a fluorescence turn-on assay for alkaline phosphatase (ALP) and creatine kinase (CK) is proposed, by using AMP and ADP as the substrate, respectively. Specifically, the GO-quenched P system containing substrate shows low fluorescence intensity. In the presence of target enzyme, the substrate is converted into either AD or ATP which have higher affinity with P, resulting in stronger fluorescence of the mixture of P and GO. The entire assay is sensitive and selective. More importantly, the ability of GO with suitable concentration to improve the specificity of aptamers not only offers an exciting new way to detect protease, but also is valuable for developing the application of GO and aptamers in the biosensing field and is expected to be used in aptamer screening systems, to improve the specificity of screened aptamers.