Development Of An Electrochemical DNA Sensor For The Detection Of α-thalassemia And β-thalassemia

Background:Thalassemias (referred to as thalassemia) are now the most common autosomal recessive blood disorders in the world, is due to globin gene defects, resulting globin chain synthesis reduced or absent, so that the formation of hemoglobin chain/non-chain imbalance, caused by a group of serious threat to human healthy death,disabling inherited blood disease. People with thalassaemia are not able to make enough haemoglobin, which is found in red blood cells. When there is not enough haemoglobin in the red blood cells, oxygen cannot get to all parts of the body. Organs then become starved for oxygen and are unable to function properly. It occurs mainly in Mediterranean countries; Southeast Asia, Africa and southern China, a conservative estimate of thalassemia gene carriers around the world nearly 200 million people, including southern China, Guangxi.Guangdong, Hainan and other provinces. Thalassemias is one of the most In China, the provinces located in the south of the Yangtze River have a relatively high incidence of thalassemia, especially Guangdong, Guangxi, and it is estimated that the population of thalassemia in those two provinces is more than 2/5 of the population of thalassemia in china.There are two major types of thalassaemia, a and β, named for the two protein chains, which gene cluster are each located on chromosome 16p13.3 and chromosome 11pl5.5 that make up normal haemoglobin. Therefore, thalassaemia is divided into two classifications, one is a-thalassemia, usually caused by the deletion or mutation of the a-globin gene (HBA) and the other is β-thalassemia, commonly caused by point mutations in the β-globin gene(HBB). Nondeletional a-thalassemia,the mutation of a-globin gene,induced by point mutation of the a2 gene (Constandspringmutation[CS],Quonszemutation[QS],and Weastmead mutation[WS]), however, could be a more severe expression. In an investigation of 59 cases of HbH diseases from Guangxi, China,27 cases (45.8%) were confirmed to be nondeletional. β-thalassemia, mostly caused by point mutations of β-globin gene, is a subset of the β-hemoglobinopathies characterized by a hereditary anemia with a wide phenotypic spectrum that can have significant morbidity and mortality. To date, over 20 β-thalassemia mutations have been identified in the Chinese population, and four mutations [CD41-42(-CTTT), IVS-Ⅱ-654(C＞T), CD17(AAG>TAG), and-28(A>G)] account for approximately 90% of the cases.Populations in South China have high frequencies of thalassemia caused by a or β globin gene mutations and/or deletions. Thus, Thalassemia is now not only an important public health problem but also a socio-economic problem of southern China.The most cost-effective strategy for reducing the burden of haemoglobin disorders like thalassaemias is to complement disease management with prevention programmes. To decrease the birth rate of critically ill patients, genotyping is necessary to identify silent carriers and thalassemia minor individuals, especially in premarital health examinations. The classic methods of detection include dot-blot and reverse dot-blot with allele-specific oligonucleotide probes, amplification refractory mutation system, multiplex amplification refractory mutation system and direct DNA sequencing. However,with such relatively complex, time-consuming, and cumbersome procedures,They are not practical and suitable for rapid screening of thalassemia at-risk populations.Biosensor is a new and developing detection technique for that combining biochemical, electrochemical, medical and electronic techniques. Recently, electrochemical DNA sensor has received a great deal of interest in many fields due to its unique advantages, such as an innately high sensitivity, simple instrumentation, automation and low cost, including detection of infectious diseases, genetic diagnosis, environmental pollutant determination,food safety, epidemiological studies, forensic identification and detection diagnosis of clinical pathogenic microorganism. The gold electrode of the electrochemical DNA sensor is functionalised with DNA probe sequence, the oligophenylmethyl molecular wires, and polyethylene glycol insulator molecules. The target is captured on electrode and it hybridises to second reporter sequence, labelled with ferrocene which we called signal probe. 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.Electrochemical DNA sensor is a novel and developing technique that combining nucleic acid hybridization and electronic techniques with the advantages of being simple, reliable, cheap, sensitive and selective for genetic detection, and has been a hot tonic in the field of biochemistry and medicine. The electrochemical signal was recorded when the target DNA hybridizes to the capture probe and ferrocene-labeled signal probe, thereby bringing the reporter molecule, ferrocene, into contact with the self-assembled monolayer on the gold electrode. Analysis can be realized high-through, rapidly, sensitively and efficient in many sample, electrochemical DNA sensor can be also widely used in the analysis of gene expression, gene diagnosis, the study and explore of new SNPs, genome and DNA sequencing.As a model for the development and validation of the method, we have developed an electrochemical DNA sensor capable of rapid detecting of thalassemia. we detected 3 of nondeletional a-thalassemia mutations and 12 of the most common β-thalassemia sequence variants that constitute more than 90% of all thalassemia mutation determinants found China.The mutations analyzed include αCS,αQS,αWS,-28(A>G), Cap+1(A>C), Int(ATG>AGG), CD14-15(+G), CD17(AAG>TAG),CD26 (GAG>AA G),CD27-28(+C),CD31(-C), CD41-42(-CTTT),CD43(G>T),CD71-72(+A) and IVS-II-654(CT). To provide a whole new method for a-thalassemia and β-thalassemia clinical auxiliary diagnosis.Method:Ⅰ. The construction of thalassemia electrochemical DNA sensor and majorization of testing detection1. Design and synthesis of DNA capture probes and signaling probes. For these experiments, we designed capture and signal probes to hybridize specifically to HBA and HBB sequences within the region amplified by the HBA and HBB primer set for the 3 of nondeletional a-thalassemia mutations and 12 of the β-thalassemia sequence variants.2. Design and synthesis of electrochemical hybrid templates, we design and synthesis the sequences which are completely complementary to the capture and signal probes as hybrid templates for the testing of thalassemia electrochemical DNA sensor.3. Optimization of the signal probes concentration. After the fast and damage-free plasma processing on the surface of the exposed gold electrodes, 6pmol/L S-S-ssDNA capture probes are deposited as a mixed solution of with the other components to form a self-assembled monolayer (SAM). After deposition, DNA sensors are rinsed, dried, and glued into a plastic housing with a port for sample introduction for hybridization. Fixed hybrid template concentration, concentratio of the signal probes were 1.5pmol/L,2.5pmol/L,3.5pmol/L,4.5pmol/L, 5.5pmol/L,6.5pmol/L,7.5pmol/L,8.5pmol/L,9.5pmol/L and 10.5pmol/L in trun. Using a series of concentration gradients experiments, the performance of sensor was assessed by cyclic voltammetry to investigate the detection range of the sensor for the optimization of signal probes concentration.4. Optimization of the hybridization time. Electrochemical hybrid template is then added to the cartridge of the DNA sensor after capture probes deposition, mixed with a signal probe cocktail. The hybridization time were set 15min,20min,25min, 30min,35min,40min,45min and 60min in trun. Using a series of time gradients experiments, the performance of sensor was assessed by cyclic voltammetry to investigate the detection range of the sensor for the optimization of the hybridization time.5. Optimization of the hybridization temperature. According to the Tm(about 45 ℃) value of the capture and signal probes, the hybridization temperature were set from 40℃ to 47℃,8 temperature gradients. Using a series of hybridization temperature gradients experiments, the performance of sensor was assessed by cyclic voltammetry to investigate the detection range of the sensor for the optimization of the hybridization temperature.Ⅱ. The establishment of the PCR detection method of thalassemia electrochemical DNA sensor1. Design and synthesis of Primers. Four pairs of primers were designed by Primer Premier 5.0 and Oligo 6 for multiplex asymmetric PCR that produces 1 fragments of the HBA gene by HBA-F and HBA-R,3 fragments of the HBB gene by HBB-1 Fand HBB-1R,HBB-2Fand HBB-2R,HBB-3F and HBB-3R, flanking all 12 mutations of interest.2. Optimization of the multiple asymmetric PCR amplification system.Using the wildtype human genome DNA as samples, from the single symmetry PCR amplification, the single asymmetric PCR amplification to multiple asymmetric PCR amplification in turn to test the optimization of primer dosage with the outcome of electrophoresis and electrochemical detection signal value.Ⅲ. Evaluation of the thalassemia electrochemical DNA sensor performance901 cases blood samples confirmed by gene sequencing,were testd by the established detection method of thalassemia electrochemical DNA sensor for qualitative detection for the validation of the ability and reliability of thalassemia electrochemical DNA sensor.Result:Ⅰ. The construction of thalassemia electrochemical DNA sensor and majorization of testing detectionCapture probes,signal probes and the electrochemical hybrid templates were synthesized by Sangon Biotech(shanghai). Fixed the capture probes concentration at 6pmol/L and hybrid template concentration, The experimental results can be seen the redox peak current didn’t increase until the concentration was more than 3.5pmol/L. When the concentration up to 5.5pmol/L,response current of redox peak current increased dramatically.It tended to increased in a linear value when the concentration was between 3.5pmol/L and 8.5pmol/L.It tended to keep in a certain stable value when the concentration up to 8.5pmol/L. Within 45min, the electrochemical signal value showed a trend of increase with the increase of the hybridization time. It tended to increased smoothly when the hybridization time between 30min and 45min. Based on the best balance of detection time and detection signal value, we choose 30min as the optimization for the hybridization time. When the hybridization temperature at 40 ℃,41 ℃ and 42 ℃ condition, the nonspecific signals are high which is not conducive to the judgment. And at 44 ℃-47 ℃ condition, although almost without the nonspecific signals, but the signal value was also reduce very obviously. Ultimately determine 43 ℃ as the optimization for the hybrid temperature.Ⅱ. The establishment of the PCR detection method of thalassemia electrochemical DNA sensorPrimers were synthesized by Sangon Biotech(shanghai).The multiplex asymmetric PCR was performed in a 50 μL-reaction consisting of 10× buffer (TAKARA) 5μL,0.2μL of lOpmol/L primers HBA-F,0.1 μL of 10pmol/L primers HBB-1F,HBB-2F and HBB-3F, 1μL of lOpmol/L primer HBA-R, HBB-1R,HBB-2R and HBB-3R, dNTPs (25mmol/L) 0.4μL,Betaine(5mmol/L) 8μL,3.0 U HotStarTaq polymerase, and 2μL of DNA template. The reaction was carried out with initial denaturation at 94℃for 15 min,followed by 35 cycles of denaturation at 94 ℃ for 45 seconds, annealing at 58℃ for 30 seconds and extension at 72 ℃ for 45 seconds,10 min　at　72℃.Ⅲ. Evaluation of the thalassemia electrochemical DNA sensor performanceApplication of 901 cases of clinical samples to verify the electrochemical DNA sensor features,15 kinds of thalassemia point mutation sites can be accurately identified. When the pairing between the target DNA and ferrocene signal probe was fully complementary, response current of redox peak current increased dramatically after hybridization of target sequence. 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 thalassemia electrochemical DNA sensor was high specific.Conclusion:Through the optimization of reaction conditions, S-S-ssDNA as capture probes, ferrocene modified signal probes, based on the principle of complementary base pairing, using electrochemical DNA sensor method to construct the thalassemia classification reagent for point mutations genotyping, have realistic feasibility.The electrochemical DNA sensors can accurately identify the mismatch of single-base in a certain concentration range.The thalassemia electrochemical DNA sensor are expected to be used in production by further optimization. With the advantage of specificity, sensitivity, and easy operation, the thalassemia electrochemical DNA sensor will clinical practice and promotion easily.