Study Of Novel Gold Nanoparticle-based Biosensors

A biosensor is a device using biological materials (eg. enzymes, antibodies, nucleic acids, microorganisms, cells, etc) as sensing elements for the detection of biological active molecules. Because of the high specificity and sensitivity, it is widely applied in medical & health, biotechnology, and environmental protection. Gold nanoparticles, due to their unique properties, are able to improve the function of a biosensor when incorporated with biological elements. Therefore, gold nanoparticle-based biosensors have received more and more attention in recent years.1. A new strategy for a DNA assay based on target triggered isothermal exponential degradation reactionDetection of specific nucleic acid sequences is essential for clinical identification of pathogens and early detection of cancers. We herein propose an ultra-sensitive, fast, simple and convenient-operated method for sequence-specific polynucleotides detection. The method is based on a novel target triggered isothermal exponential degradation reaction (TT-isoTexpDR), which integrates polymerase strand extension and double strand cleavage. In the presence of target DNA (fM level), linker strand (100 nM) can be degraded by TT-isoTexpDR in 8.5 minutes and the degradation can be further distinguished by probe functionalized gold nanoparticles within minutes. The total time for DNA detection is only 15 minutes. In addition to high sensitivity and efficiency, this assay also shows great specificity that is able to discriminate one base mismatch.2. Highly sensitive aptasensor for potassium detection fabricated with a nicking endonuclease-assisted signal amplification strategyA novel strategy to fabricate 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. 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 naked eyes. Results show that 2μL sample containing 0.1 mM of potassium is enough to induce distinct color appearance of the nanoparticles, and potassium ion can be easily distinguished from many other ions.3. Study of colorimetric assay for triplex DNATriplex DNA technology has been considered as an attractive antigene strategy for the treatment of genetic-based diseases. Assay of the formation of triplex is an important part in the development of triplex technology. In this paper, we present a novel method to assay triplex DNA, based on the unspecific interaction between single stranded triplex-forming oligonucleotide and negatively charged gold nanoparticles. While triplex is formed, gold nanoparticles will aggregate without the protection of triplex-forming oligonucleotide under a certain concentration of salt. Consequently, the color of the gold nanoparticles will change from red to blue. The formation of triplex DNA and the discrimination of triplex-forming oligonucleotide candidates are thereby easily monitored by the color changes of gold nanoparticles. Also by precisely controlling the working salt concentration, we are allowed to assay single-nucleotide polymorphism of triplex-forming oligonucleotides. Mismatched variants and length variants of triplex-forming oligonucleotides with single-nucleotide or double-nucleotides differences can be well discriminated. This method presented here is simple, fast, and with considerable selectivity, so we expect it will be a promising candidate for the assay of triplex DNA and the screening of appropriate triplex-forming oligonucleotide.4. A novel electrochemical method to detect mercury (Ⅱ) ions based on gold nanoparticles-assisted signal amplificationA novel and sensitive electrochemical method for determination of mercury (Ⅱ) ions (Hg2+) based on the formation of thymine-Hg2+-thymine complexes and gold nanoparticle-mediated signal amplification is reported. Two 5'end thiolated complementary oligonucleotides containing six strategically placed thymine-thymine mistakes are introduced in this work. One of the two oligonucleotides is immobilized on a gold electrode and the other one on gold nanoparticles (Au NPs). Due to six thymine-thymine mistakes, the two oligonucleotides are not able to be hybridized, so Au NPs can not be immobilized onto the electrode surface after the DNA modified electrode is immersed in the DNA-Au NPs solution. However, if Hg2+ exists, T-Hg2+-T complexes will be formed and Au NPs can be immobilized onto the electrode surface. Meanwhile, large numbers of [Ru(NH3)6]3+ molecules as electrochemical species will be localized onto the electrode surface. The Hg2+ detection limit of this assay can be as low as 10 nM, which is the U.S. Environmental Protection Agency (EPA) limit of Hg2+ for drinkable water. This method is proven to be simple, convenient, high sensitive and selective.5. Fabrication of multi-functionalized gold nanoparticles and the application to electrochemical detection of nitriteThis paper reports the fabrication of multi-functionalized gold nanoparticles (MFAuNPs) and the application to electrochemical detection of nitrite. While gold nanoparticles are modified with thiolated oligonucleotides as usual, they are also immobilized with 5-[1,2] dithiolan-3-yl-pentanoic acid [2-(naphthalene-1-ylamino)-ethyl]amide (DPAN). Therefore, the oligonucleotides molecules can enhance the solubility of the MFAuNPs and absorb hexaammineruthenium (Ⅲ) chloride ([Ru(NH3)6]3+) as electrochemical species on the one hand, in the presence of nitrite ions, DPAN immobilized on MFAuNPs will react with 4-(2-aminoethyl)benzenamine which has been previously modified on the surface of a gold electrode via Griess reaction on the other hand, thus electrochemical detection of nitrite can be achieved. Although the maximum contaminant level (MCL) defined by the Environmental Protection Agency (EPA) for nitrite in drinking water is as low as 1 ppm (21.7μM), which is difficult to make detections by the current techniques, the proposed method in this work can give very satisfactory results.6. Electrochemical detection of DNA based on exonuclease and gold nanoparticlas-assisted dual signal amplificationHerein we report a novel, sensitive and convenient electrochemical method for DNA detection by using the exonucleaseⅢ(exoⅢ) and probe DNA P2 functionalized gold nanoparticals (P2-Au NPs). Probe DNA P1 on the surface of gold electrode can form a rigid stem-loop structure with 5 non-complementary bases in 3'termini, so P1 can neither be digested by ExoⅢnor hybridize with P2-Au NPs. In the presence of target DNA, P1 will hybridize with target DNA and consequently be digested by exoⅢfrom its 3'-hydroxyl termini to the end of the duplex. With the target DNA cycling, multiple P1 will be digested by ExoⅢand transformed from the stem-loop conformation to a flexible linear structure. Then, the digested P1 can hybridize with P2-Au NPs and result in an amplified electrochemical response. The results have shown that this electrochemical biosensor based on dual signal amplification can detect target DNA with an improved sensitivity and selectivity. The linear range is from 100 pM to 10 nM with the detection limit of 33 pM.