Genxi Li

Novel method to detect DNA methylation using gold nanoparticles coupled with enzyme-linkage reactions.

DNA methylation, catalyzed by methylases, plays a critical role in many biological processes, and methylases have been regarded as promising targets for antimicrobial drugs. In this paper, we propose a simple and sensitive colorimetric assay method to detect the activity of methylases so as to monitor DNA methylation using DNA-modified gold nanoparticles (AuNPs) coupled with enzyme-linkage reactions. The duplex DNA molecules modified on the surface of AuNPs are first methylated by DNA adenine methylation (Dam) methyltransferase (MTase) and then cut by methylation-sensitive restriction endonuclease Dpn I. Removal of duplex from the AuNP surfaces by the methylation/cleavage process will destabilize the nanoparticles, resulting in aggregation of AuNPs and a red-to-blue color change. Consequently, the enzyme activity of Dam MTase can be assayed and DNA methylation can be detected. Furthermore, this study may provide a sensitive platform to screen inhibitors for Dam MTase.

Protein Detection Based on Small Molecule-Linked DNA

Based on small molecule-linked DNA and the nicking endonuclease-assisted amplification (NEA) strategy, a novel electrochemical method for protein detection is proposed in this work. Specifically, the small molecule-linked DNA (probe 1) can be protected from exonuclease-catalyzed digestion upon binding to the protein target of the small molecule, so the DNA strand may hybridize with another DNA strand (probe 2) that is previously immobilized onto an electrode surface. Consequently, the NEA process is triggered, resulting in continuous removal of the DNA strands from the electrode surface, and the blocking effect against the electrochemical species [Fe(CN)(6)](3-/4-) becomes increasingly lower; thus, increased electrochemical waves can be achieved. Because the whole process is activated by the target protein, an electrochemical method for protein quantification is developed. Taking folate receptor (FR) as an example in this work, we can determine the protein in a linear range from 0.3 to 15 ng/mL with a detection limit of 0.19 ng/mL. Furthermore, because the method can be used for the assay of FR in serum samples and for the detection of other proteins such as streptavidin by simply changing the small molecule moiety of the DNA probes, this novel method is expected to have great potential applications in the future.

Effect of silver nanoparticles on the electron transfer reactivity and the catalytic activity of myoglobin.

Silver nanoparticles (11±1.5 nm) could greatly enhance the electron‐transfer reactivity of myoglobin (Mb) and its catalytic ability toward hydrogen peroxide (H2O2). Direct fast electron transfer between Mb and a pyrolytic graphite (PG) electrode was achieved, and a pair of well‐defined, quasireversible redox peaks was obtained. The cathodic and anodic peaks were located at −329 and −281 mV, respectively. Meanwhile, the catalytic ability of the protein toward the reduction of H2O2 was also studied, and a H2O2 biosensor was subsequently fabricated. Its detection limit was 1.0×10−6 M with a sensitivity of 0.0205 μA per μM of H2O2. The apparent Michaelis–Menten constant was calculated to be 1303 μM. Flocculation assay showed that the protein maintained plasmon layers surrounding the surface of silver nanoparticles and avoided silver‐nanoparticle aggregation. On the other hand, UV‐visible spectroscopy studies revealed that silver nanoparticles could induce a small change of the heme‐group environment of the protein; this contributed to the enhancement of the electron‐transfer reactivity and the catalytic activity.

A reagentless nitric oxide biosensor based on hemoglobin–DNA films

Nitric oxide (NO) is an important bioregulatory molecule in vivo. A reagentless, hemoglobin (Hb) based nitric oxide biosensor is prepared, based on the direct electrochemistry of Hb. Spectroscopic studies show that Hb maintains most of its 3-dimensional structure in the film, and keeps its reactivity with NO. The electrochemical reduction of NO is catalyzed employing the Hb modified electrode. Experimental results reveal that the peak current related to NO is linearly proportional to its concentration. It can detect traces of NO as low as 2.9 μM. This biosensor is also shown to be simply constructed, biocompatible and stable.

Direct Electrochemistry and Enhanced Catalytic Activity for Hemoglobin in a Sodium Montmorillonite Film

Direct electrochemistry of hemoglobin is obtained by coimmobilizing hemoglobin and sodium montmorillonite at a pyrolytic electrode surface. Hemoglobin maintains most of its native structure in the film. This modified electrode shows high stability and enzyme-like catalytic activity.

Fabrication of a Highly Sensitive Aptasensor for Potassium with a Nicking Endonuclease-Assisted Signal Amplification Strategy

A novel strategy to fabricate an aptasensor for potassium with high sensitivity and selectivity by using nicking endonuclease is proposed in this work. A nicking endonuclease (Nt.CviPII), which may recognize specific nucleotide sequences in double-stranded DNA formed by a potassium-binding aptamer and a linker DNA but cleave only the linker strand, may transfer and amplify the quantitative information of the potassium detection to that of the linker DNA through elaborate strand-scission cycles. Since the technique for gene assay is much more mature, the linker DNA can thereby be detected by a number of available methods. Here, taking advantage of a simple and fast gold nanoparticles-based sensing technique, we are able to assay the linker and consequently potassium ion simply by UV-vis spectroanalysis and even with the naked eye. Results show that a 2 μL sample containing 0.1 mM of potassium is enough to induce distinct color appearance of the nanoparticles, and the potassium ion can be easily distinguished from many other ions. The strategy proposed in this work shows some unique advantages over some traditional methods and may be further developed for the detection of some other chemicals in the future.

Strategy to fabricate an electrochemical aptasensor: application to the assay of adenosine deaminase activity.

A novel strategy for the fabrication of electrochemical aptasensor is proposed in this work, and the strategy has been employed to develop an aptasensor for the assay of adenosine deaminase activity. While a well-designed oligonucleotide containing three functional regions (an adenosine aptamer region, a G-quadruplex halves region, and a linker region) is adopted in our strategy as the core element, the enzymatic reaction of adenosine catalyzed by adenosine deaminase plays a key role as well in the regulation of the binding of the G-quadruplex halves with hemin, the electroactive probe, which is to reflect the activity of the enzyme indirectly but accurately. The detection limit of the fabrication biosensor can be lowered to 0.2 U mL(-1) of adenosine deaminase, and 1 nM of the inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride is enough to present distinguishable electrochemical response. Moreover, since the electroactive probe is not required to be bound with the oligonucleotide, this strategy may integrate the advantages of both the labeled and label-free strategies.

Detection of breast cancer cells specially and accurately by an electrochemical method.

Breast cancer is one of the most common cancers to cause death in the world, and the accurate diagnosis is of great importance to determine the stage of the disease and then to design the suitable therapy. Compared with the traditional detection methods relying on the recognition of only one tumor marker, we herein propose a sensitive electrochemical immunoassay to detect breast cancer cells by simultaneously measuring two co-expressing tumor markers, human mucin-1 and carcinoembryonic antigen on the surface of the cancer cells, which may efficiently improve the accuracy of the detection as well as facilitate the classification of the cancer cells. The experimental results have revealed that well electrochemical response can be observed only under the condition that both of the tumor markers are identified on the surface of the tumor cells. With this method, breast cancer cell MCF-7 can be easily distinguished from other kinds of cells, such as acute leukemia cells CCRF-CEM and normal cells islet beta cells. Moreover, the prepared cytosensor can specially monitor breast cancer cell MCF-7 in a wide range from 10(4) to 10(7) cell mL(-1) with well reproduction and low detection limit, which may have great potential in clinical applications.

Electrochemical Approach To Detect Apoptosis

This paper reports an electrochemical approach for detection of apoptosis. Here we prepare a gold electrode modified with a helix peptide ferrocene (Fc)-GDGDEVDGC. Fc is used as an electroactive reporter and the peptide as a recognition and cleavage site of caspase-3, which is a special proteinase to apoptosis. Results show that this method may sensitively and specifically detect apoptotic cells with signal decline of 85%. This approach is different from the previous methods for apoptosis detection, because it does not need any fluorescent materials, expensive biological instruments, or complicated procedures.

A nitric oxide biosensor based on the multi-assembly of hemoglobin/montmorillonite/polyvinyl alcohol at a pyrolytic graphite electrode.

Direct electron transfer of hemoglobin (Hb) can be achieved in a Hb/montmorillonite (MMT)/polyvinyl alcohol multi-assembly at a pyrolytic graphite (PG) electrode. Accordingly, a novel nitric oxide (NO) biosensor is proposed. The reduction of NO is observed at a potential of approximately -783 mV (vs. SCE) at pH 5.5. At optimum pH, this biosensor shows a wide linear range of 1.0x10(-6)-2.5x10(-4) mol/l with a detection limit of 5.0x10(-7) mol/l. The sensor-to-sensor reproducibility is good consideringmacr; a relative standard deviation of 3.5% in five independent determinations at 5.0x10(-5) mol/l NO. The modified electrode is conveniently constructed and durable in long-term operations.