New Detection Methods Of Urinary Adenosine Based On Malachite Green-nucleic Acid Probe

Urinary adenosine (AD) could be used as a potential biomaeker for cancer andother diseases. Some studies found that the rapid growth of tumor cells could resultin the degradation of adenine nucleotide, leading to the increase of the productionand discharge of adenosine. This promoted the rapid growth of tumor, making theadenosine being accumulated in body by the process. In addition, adenosine is asignificant active endogenous substance and plays an important role in peripheralnervous system, central nervous system, immune system and cardiovascular system.Therefore, the detection of urinary adenosine is of practical significance in earlydiagnosis and prognosis monitoring of malignant tumor.In the chapter2, a resonance light scattering method for the detection ofurinary adenosine has been developed by using a malachite green as a reporter andaptamer as a probe. The nucleic acid prob was designed to fold into a hairpinstructure. In the presence of adenosine, it could combine with the aptamer part of NP,resulting in the conformational change of NP to form a G-quadruplex with a part ofsingle stranded DNA (ssDNA). It could combine with malachite green (MG), leadingto the increase of RLS intensity of this system. Under the optimal conditions, The ΔIis directly proportional to the concentration of AD in the range of7.5×10-11~2.2×10-9mol·L-1. The equation of linear regression is I=18.9c (×10-10mol·L-1)+16.5with a correlation coefficient of r=0.9975. The limit of detection (LOD) was2.3×10-11mol·L-1. The method is simple, sensitive, and has been used in thedetection of AD in the urine samples.In the chapter3, a spectrophotometric mothod for AD in human urine has beenestablished based on the conformational change of nucleic acid probe to form aG-quadruplex with a part of single stranded DNA (ssDNA). The resulting structurecould interact with MG, resulting in the enhancement of absorbance of this system. A/A0linearly correlated with the concentration of AD over the ranges of5.2×10-10~1.8×10-8mol·L-1. The equation of linear regression is A/A0=1.04c (×107mol·L1)+1.04with a correlation coefficient of r=0.9976. The limit of detection (LOD) was1.6×10-10mol·L-1. The proposed method without the requirement of expensiveequipment and pretreatment of samples, is simple, rapid, selective, and has been usedin the detection of AD in the urine with a satisfactory result.In the chapter4, the possible secondary structures of nucleic acid probe in theabsence of AD were predicted using the Primer Premier6.0program andRNAstructure, which was demonstrated to fold into a hairpin structure. Theinteraction mechanism among nucleic acid probe, adenosine and malachite greenwas investigated by using gold nanoparticles colorimetric method, TMB-AuNPs-Hg2+mimic enzyme, NMM resonance light scattering and fluorescence method, andcircular dichroism spectra. The experimental results confirmed our presumption,which paves the theory basis, and provides experimental evidences for expanding theapplication of nucleic acid probe.