Dun Pan

Visual Cocaine Detection with Gold Nanoparticles and Rationally Engineered Aptamer Structures

A novel bioassay strategy is designed to detect small-molecule targets such as cocaine, potassium, and adenosine, based on gold nanoparticles (AuNPs) and engineered DNA aptamers. In this design, an aptamer is engineered to be two pieces of random, coil-like single-stranded DNA, which reassembles into the intact aptamer tertiary structure in the presence of the specific target. AuNPs can effectively differentiate between these two states via their characteristic surface-plasmon resonance-based color change. Using this method, cocaine in the low-micromolar range is selectively detected within minutes. This strategy is also shown to be generic and applicable to the detection of several other small-molecule targets.

Fluorescent In Situ Targeting Probes for Rapid Imaging of Ovarian‐Cancer‐Specific γ‐Glutamyltranspeptidase

γ-Glutamyltranspeptidase (GGT) is a tumor biomarker that selectively catalyzes the cleavage of glutamate overexpressed on the plasma membrane of tumor cells. Here, we developed two novel fluorescent in situ targeting (FIST) probes that specifically target GGT in tumor cells, which comprise 1) a GGT-specific substrate unit (GSH), and 2) a boron-dipyrromethene (BODIPY) moiety for fluorescent signalling. In the presence of GGT, sulfur-substituted BODIPY was converted to amino-substituted BODIPY, resulting in dramatic fluorescence variations. By exploiting this enzyme-triggered photophysical property, we employed these FIST probes to monitor the GGT activity in living cells, which showed remarkable differentiation between ovarian cancer cells and normal cells. These probes represent two first-generation chemodosimeters featuring enzyme-mediated rapid, irreversible aromatic hydrocarbon transfer between the sulfur and nitrogen atoms accompanied by switching of photophysical properties.

Gold nanoparticles for high-throughput genotyping of long-range haplotypes

Completion of the Human Genome Project and the HapMap Project has led to increasing demands for mapping complex traits in humans to understand the aetiology of diseases. Identifying variations in the DNA sequence, which affect how we develop disease and respond to pathogens and drugs, is important for this purpose, but it is difficult to identify these variations in large sample sets. Here we show that through a combination of capillary sequencing and polymerase chain reaction assisted by gold nanoparticles, it is possible to identify several DNA variations that are associated with age-related macular degeneration and psoriasis on significant regions of human genomic DNA. Our method is accurate and promising for large-scale and high-throughput genetic analysis of susceptibility towards disease and drug resistance.

Gold nanoparticlebased optical probes for target-responsive DNA structures

In this work, we report the use of unmodified gold nanoparticles (AuNPs) as an optical probe for the detection of target-responsive structural variations of DNA. By employing two DNA structures, i.e., a pH-responsive i-motif oligonucleotide and a mercury-specific oligonucleotide (MSO), we demonstrated that AuNPs could selectively distinguish target-free and target-bound oligonucleotides via the characteristic surface plasmon resonance-associated red-to-blue color change. Based on these observations, we developed a convenient “mix-and-detect” approach that could selectively detect environmentally toxic mercury ions.

DNA-conjugated quantum dot nanoprobe for high-sensitivity fluorescent detection of DNA and micro-RNA.

Herein, we report a convenient approach to developing quantum dots (QDs)-based nanosensors for DNA and micro-RNA (miRNA) detection. The DNA-QDs conjugate was prepared by a ligand-exchange method. Thiol-labeled ssDNA is directly attached to the QD surface, leading to highly water-dispersible nanoconjugates. The DNA-QDs conjugate has the advantages of the excellent optical properties of QDs and well-controlled recognition properties of DNA and can be used as a nanoprobe to construct a nanosensor for nucleic acid detection. With the addition of a target nucleic acid sequence, the fluorescence intensity of QDs was quenched by an organic quencher (BHQ2) via Förster resonance energy transfer. This nanosensor can detect as low as 1 fM DNA and 10 fM miRNA. Moreover, the QDs-based nanosensor exhibited excellent selectivity. It not only can effectively distinguish single-base-mismatched and random nucleic sequences but also can recognize pre-miRNA and mature miRNA. Therefore, the nanosensor has high application potential for disease diagnosis and biological analysis.

Gold Nanoparticle-Based Enzyme-Linked Antibody-Aptamer Sandwich Assay for Detection of Salmonella Typhimurium

Enzyme-linked immunosorbent assay (ELISA) provides a convenient means for the detection of Salmonella enterica serovar Typhimurium (STM), which is important for rapid diagnosis of foodborne pathogens. However, conventional ELISA is limited by antibody-antigen immunoreactions and suffers from poor sensitivity and tedious sample pretreatment. Therefore, development of novel ELISA remains challenging. Herein, we designed a comprehensive strategy for rapid, sensitive, and quantitative detection of STM with high specificity by gold nanoparticle-based enzyme-linked antibody-aptamer sandwich (nano-ELAAS) method. STM was captured and preconcentrated from samples with aptamer-modified magnetic particles, followed by binding with detector antibodies. Then nanoprobes carrying a large amount of reporter antibodies and horseradish peroxidase molecules were used for colorimetric signal amplification. Under the optimized reaction conditions, the nano-ELAAS assay had a quantitative detection range from 1 × 10(3) to 1 × 10(8) CFU mL(-1), a limit of detection of 1 × 10(3) CFU mL(-1), and a selectivity of >10-fold for STM in samples containing other bacteria at higher concentration with an assay time less than 3 h. In addition, the developed nanoprobes were improved in terms of detection range and/or sensitivity when compared with two commercial enzyme-labeled antibody signal reporters. Finally, the nano-ELAAS method was demonstrated to work well in milk samples, a common source of STM contamination.

Folding super-sized DNA origami with scaffold strands from long-range PCR

A 26 kilobase single strand DNA fragment was obtained from long-range PCR amplification and subsequent enzymatic digestion, which we folded into a super-sized DNA origami nanostructure by using ∼800 staple strands.

Amplified Fluorescent Recognition of G-Quadruplex Folding with a Cationic Conjugated Polymer and DNA Intercalator

The single stranded DNA (ssDNA) with G-rich sequence can fold into G-quadruplex via intramolecular hydrogen-bonding interaction in the presence of ligand. This structure conversion can be specifically detected by a fluorescence method based on different interaction between SYBR Green I (SG) and various DNA structures. SG is proved to intercalate into G-quadruplex and results in high fluorescence intensity, which can be further amplified by 6-fold through fluorescence resonance energy transfer (FRET) from a water-soluble cationic conjugated polymer (CCP) to SG due to the high affinity of positively charged CCP to negatively charged rigid G-quadruplex, whereas it is not performed for ssDNA in the absence of K(+). As a result, the ssDNA/SG/CCP complex can be used to detect potassium ions with improved selectivity in a label-free and cost-effective manner.

Electrochemical interrogation of interactions between surface-confined DNA and methylene blue

In this work, we reported a systematic investigation on the interactions between methylene blue (MB) and surface-confined DNA by using electrochemical methods. We demonstrated that the redox potential of MB and binding and dissociation kinetics of MB to DNA differed significantly for single-stranded DNA (ss-DNA) and double-stranded DNA (ds-DNA) immobilized on gold electrodes. This was possibly due to the different binding mechanism between MB and ss- or ds-DNA. This work might provide useful information for developing MB-based sequence-specific electrochemical DNA sensors.

Modulation of DNA Polymerases with Gold Nanoparticles and their Applications in Hot-Start PCR

A new gold-nanoparticle (AuNP)-based strategy to dynamically modulate the activity of DNA polymerases and realize a hot-start (HS)-like effect in the polymerase chain reaction (PCR) is reported, which effectively prevents unwanted nonspecific amplification and improves the performance of PCRs. A high-fidelity Pfu DNA polymerase is employed as the model system. Interestingly, AuNPs inactivate the polymerase activity of Pfu at low temperature, thus resembling an antibody-based HS PCR. This inhibition effect of AuNPs is demonstrated for the preamplification polymerization activity of the PCR, which largely suppresses nonspecific amplification at temperatures between 30 and 60 degrees C and leads to highly specific and sensitive PCR amplification with Pfu. Significantly, the fidelity of Pfu is not sacrificed in the presence of AuNPs. Therefore, this AuNP-based HS strategy provides a straightforward and potentially versatile approach to realize high-performance PCR amplification.