Establishment Of Electrochemical Biosensor For Detecting CYP3A5~*3A Gene Polymorphism

BackgroundPharmacogenomics, also known as genomic medicine or the genome of pharmacology, is a new subject mainly focuses on the mechanism and phenomenon of absorption metabolism, efficacy, and adverse reactions of drugs. It aims at guiding the development of new drug and rational drug use. Genetic polymorphism is the basic of pharmacogenomics; it mainly performs as polymorphism of drug-metabolizing enzymes, transporters, receptors and drug targets. It may lead to individual differences in efficacy and side effects of certain drugs. Cytochrome P450enzymes are members of a protease superfamily whose argons are hemoglobin. They are involved in the metabolism of many physiological substances, toxic substances, carcinogens, and about90%of clinical drugs. The synthesis of CYP450is regulated by gene. CYP450enzymes and drug metabolism may change following the mutation of gene. The polymorphism of CYP450is the main reason for individual variation of drug metabolism, and CYP3A4and CYP3A5involve in the metabolism of50%of commonly used drugs. The metabolism of preferred immunosuppressant drug for clinical transplantation mainly is affected by CYP3A5gene. Individuals who carry CYP3A5*1can rapidly metabolized tacrolimus, which reduces the blood drug concentration. Therefore, it’s very important to analyze the CYP3A5gene type of patients before they take organ transplantation. It aims at achieving optimal dose, toxicity and the treatment effect, realizing the individualization of clinical treatment by dosage adjustment, and choosing the most suitable immunosuppressant combinations and doses.Gene polymorphism analysis is a hot research method in modern life science. There are many traditional methods to detect gene mutations:DNA sequencing, Taqman probe, single strand conformation polymorphism, restriction fragment length polymorphism, pyrosequencing, amplification refractory mutation system, and denaturing high performance liquid chromatography. These methods need to detecte the samples by gel electrophoresis and illusion of DNA secondary structure could easily lead to deviation of the results. Some methods are high cost, and others are complicated operation or low sensitivity, so none is favorable for the clinical test. Therefore, it is particularly important to develop an inexpensive, high sensitivity, simple and accurate method for clinical examination.Electrochemical sensor consists of two parts, identification components and conversion components. It combines the electrochemical powerful analysis with specificity of biological identification process. The main principle of this detection is the specificity recognition between identify components and determinants. It will produce a series of physical or chemical change during the process of binding, for example composite, light, electricity and so on. These changes which relate to the concentration of the test substance can be output by the conversion of the sensor. Electrochemical method is rapid, simple operated, inexpensive, high selective and sensitive, so it is suitable for on-line analysis. In addition, the electrochemical method further comprises a high degree of automation, miniaturization and integration features Many scientists have paid close attention to DNA electrochemical sensor for its advantages in detection DNA hybridization. Electrochemical method is rapidity, simple operated, inexpensive, and these advantages make it to be superior to other methods. A basic DNA transducer is designed by the immobilization of an ss-DNA probe on a transducer surface to recognize by its target DNA sequence via hybridization. The DNA duplex formed on the electrode surface is known as a hybrid. Electrochemical detection of hybridization is mainly based on the differences of the electrochemical conduct of the labels with or without double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). In addition, the use of the sandwich structure for DNA hybridization further improves the selective recognition of DNA electrochemical sensor, thus this DNA electrochemical sensor have high specificity.ObjectiveThis study aims to use electrochemical method, combined with molecular probe, to build a DNA biosensor detection system which is high sensitive, low cost, accurate, and simple operated. Then, the system can be used for detecting CYP3A5gene.Methods1. Optimizing the electrode modificationGold electrodes were modified by mercaptoethanol self-assembled. The gold electrodes immersed in the solution of synthetic HS-ssDNA, ssDNA and S-S-ssDNA under4℃for12hours. We investigated the modify efficiency and electrochemical effects using CV (cyclic voltammetry). HS-ssDNA was chosen as capture probe in the following experiment. In addition, the electrochemical character was estimated by changing the scan rate, and the scan rates were100mV/S, 200mV/S,300mV/S,400mV/S,500mV/S,600mV/S, and700mV/S.In order to analyze the effect of probe concentration, a series of gradient concentration of capture probes (HS-ssDNA) was designed. Concentration gradients:6.25μmol/L,12.5μmol/L,25μmol/L,50μmol/L,60μmol/L,80μmol/L, and100μmol/L, were investigated in the modify efficiency and electrochemical effects using CV (cyclic voltammetry). Moreover, we assessed the effect of time length for modification. We set time gradients as2h,4h,6h,8h,10h,12h, and14h, to compare the modify efficiency and electrochemical effect by cyclic voltammetry.2. The construction of electrochemical sensors and majorization of testing detectionImmobilization of capture probe HS-ssDNA was fixed on a transducer surface gold electrode by self-assembly, and then the blank sites were sealed off by MCH. Based on the principle of complementary base pairing, target DNA and the signal probe were combined with the modified electrode surface. The condition of reaction was40℃for2hours. The DNA duplex that formed on the electrode surface was known as a hybrid. The electrochemical characterization of modified electrodes was assessed in Fe (CN)63-/Fe (CN)64-base solution by cyclic voltammetry. The specificity of sensor was evaluated through detecting single-base mismatch with target DNA. Using a series of concentration and time gradients experiments, the performance of sensor was assessed in Fe (CN)63-/Fe (CN)6" base solution by cyclic voltammetry to investigate the detection range of the sensor. Concentrations of target sequence were0.01μmol/L,0.1μmol/L,1μmol/L,10μmol/L,25μmol/L,40μmol/L,50μmol/L,60μmol/L,70μmol/L,80μmol/L,90μmol/L and100μmol/L in turn. The concentrations of signal probe were1μmol/L,10μmol/L,25μmol/L,40μmol/L,50μmol/L,60μmol/L,70μmol/L,80μmol/L,90μmol/L and100μmol/L in turn. The time gradients of hybridization were0.5h,1h,1.5h and2h in turn.3. Testing of clinical samplesThe total genomic DNAs in blood samples of6patients were extracted using a DNA extracting kit. CYP3A5sequences containing A6986G locus were amplified by PCR. The length of the sequence was121bp. The PCR products were purified using a DNA purification kit. Then, the products were melted into single-stranded DNA under95℃for5minutes, to build electrochemical sensors by the principle of complementary base pairing. The modified gold electrode was immersed in the mixed solution of single-stranded DNA and signal probe at40℃for2hours. Then, DNA electrochemical sensor was characterized by cyclic voltammetry.Results1. Optimizing the electrode modificationThe results of the self-assembly electrodes showed that the modification of capture probe of HS-ssDNA and SS-ssDNA is better than ss-DNA in immobilization effects. The formation of Au-S bond made the capture probe firmly adsorbed on the surface of gold electrode and reduced non-specific adsorption. In addition, the end of-(CH2)6as the bracket for the probe thereby avoided the nucleotide attached with the electrode surface. There was no significant difference in modification efficiency between HS-ssDNA and S-S-ssDNA. However, the life span of S-S-ssDNA is longer than S-S-ssDNA, and the preparation of HS-ssDNA is easier than S-S-ssDNA electrode. So we chose HS-ssDNA as capture probe in the following studies. Concentration gradient experiment showed that best modification efficiency of capture probe concentration was80μmol/L. The potential and current of peaks was tending towards stability when the immobilized time was extended to12hours. So we chose12hours as the modification time of capture probe. 2. The construction of electrochemical sensors and majorization of testing detectionWhen the pairing between the target DNA and ferrocene signal probe was fully complementary, response current of redox peak current increased dramatically. The current of the redox peak changed more than30μA after hybridization of target sequence. It showed that the design of CYP3A5*3A sensor was feasible. The response current of redox peak current showed no increase even lower when there was single-base mismatch between the target DNA and ferrocene signal probe. It suggested that the sensor was high specific.The experimental results can be seen the redox peak current didn’t increase until the concentration was more than25μmol/L. It tended to keep in a certain stable value when the concentration was up to80μmol/L. The same situation also appeared in ferrocene signal probe.In order to study the property of response for current, the effects of different scan rate on peak current were investigated. The relationship between current and voltage was linear when the scant rate ranged from100mV/S to400mV/S. The response signal began to drift when the scant rate was greater than300mV/S. The results showed that the redox process of electroactive material had characteristic of surface reaction.3. Testing of clinical samplesThe purity and integrity of genomic DNA in blood samples of6patients was detected. The results of2%agarose gel electrophoresis indicated that the structural of DNA was integrated. The concentration of genomic DNA was ideal. The PCR products were in high purity and specificity, and the sequence length was121bp. The results of sequencing showed that the PCR amplification product contained the locus fragment of A6986G. The concentration of PCR product after purification was determined by UV spectrophotometer. Then, the concentration raised to1831ng/μL and the A260/A280ratio of was in the range of1.75-1.90.The characteristics of CYP3A5*3A electrochemical sensor was verified with the genomic DNA in blood samples of6patients. The results of DNA biosensors were consistent with DNA sequencing in CYP3A5gene detection. Accordingly, the CYP3A5*3A electrochemical sensor has a good specificity to distinguish genotypes.ConclusionThe SAM molecular layer is arranged in an orderly direction and facilitates the hybridization of DNA. The CYP3A5*3A DNA electrochemical sensor was built by the sandwich method, using HS-ssDNA as capture probes and ferrocene signal probe as indicator. The DNA biosensors can accurately identify the mismatch of single-base in a certain concentration range. With the advantage of specificity, sensitivity, and easy operation, DNA electrochemical biosensor will be applied in clinical practice and promotion easily.