Electrochemical Biosensor Based On In-situ Labeling Signal Tag On The Bio-interface Of DNA

With the deeper understanding of the nucleic acids, the biosensor based on DNA as bio-recognition element had been great developed. Due to the advantages of good selectivity, high sensitivity, simple operability and stable, the electrochemical biosensor based on labeling signal tag onto the interface of DNA had been achieved a wide range of applification in many aears, such as biomedical research, clinical test. However, there were still some disadvantages for this technology, for example, the processes of labeling the tag and the separation of fabricating the probe were often complicated, or it took much time and material to prepare the signal marker. In order to overcome these disadvantages, in this paper, the method of construction of biosensor will be used by in-situ labeling electroactive signal marker onto the terminal of DNA, and the fabricated biosensor will be developed to detect target DNA or heavy metal ions(lead ions). The main contents were as follows:(1) In this paper, we constructed a new kind of electrochemical biosensor based on in-situ labeling 4’-Aminobenzo-18-Crown-6-copper complexes(AC-Cu2+) on the free terminal of the molecule beacons(MBs). The MB with bifunctional groups of 3’-SH and 5’-COOH was immobilized onto the electrode via sulfur-gold affinity. Then 4’-Aminobenzo-18-crown-6(AC) was anchored on the 5’ terminal of MB through amide bond, which was used as a platform molecule for the following coordination assembly of copper ions(Cu2+). Differential pulse voltammetry(DPV) was used to detect the hybridization between biosensor and complementary strand DNA of different consistencies(S2). The results of experiment were showed that the the peak curret of the biosensor(Ipa) exhibited an excellent correlation to the logarithm of the concentration of target DNA(lg CS2) in the range of 0.1 pmol L-1 and 0.5 nmol L-1. The linear equation was Ipa(10-6 A)=1.086 +0.294 lg(CS2/ mol L-1), the correlation coefficient r was 0.993, and the detection was limited was at 61 fmol L-1, which suggested that the DNA biosensor could quantitatively analyze specific target sequence. Furthermore, hybridization specificity experiments showd that the biosensor had good selectivity to complete complementary, one-base mismatched sequence and non-complementary sequences.(2) This paper describes a novel electrochemical DNA biosensor based on in-situ labeling gold nanoparticle(Au NPs) and Neutral Red(NR), in turn, at the terminal of the molecular beacons(MBs). Because of the high specific surface area of Au NPs and the excellent electroactive of NR, the gene segment of Cauliflower mosaic virus 35 s promoter had been detected by using the electrochemical biosensor. The hybridization experiments showed that the proposed sensor exhibited good specificity for target DNA in the kinetic range from 1 fmol L-1~1 nmol L-1 with a detection limit of 0.11 fmol L-1. In addition, owing to the special loop-stem structure of DNA, the biosensor can well distinguish the target DNA from the different mismatch-complementary sequences.(3) The developed sensor was fabricated by using the Au NPs as a platform to amplify the signal, which was anchored on the 5’-terminus of the Pb2+-aptamer through the bond of Au-S, and by using the special affinity between cytosine and silver ion(Ag+) to in-situ generating the silver nanoparticles(Ag NPs) based on the DNA as template and through the reducing action of sodium borohydride(Na BH4). The presence of Pb2+ reduces the cross-reaction between lead ion-aptamer and the complementary strand, resulting that the signal-off. The signal of Ag NPs was measured by differential pulse voltammetry, while the Ag NPs@C-rich/Au NPs/S2 conjugate enables the detection of Pb2+ in the linear range of 1 pmol L-1 to 10 nmol L-1 with a detection limit that corresponded to 0.57 pmol L-1. Meanwhile, this process revealed selectivity and specificity of Pb2+ ions, even with interference by high concentration of other metal ions.