Key Words】 thymidine kinase quality zoom piezoelectric immunosensor clinical diagnosis
Thymidine kinase (thymidine kinase, TK) is important in the process of DNA synthesis kinase, is closely related to cell division and proliferation. There are two major isozymes TK, TK1 present in the cytoplasm, and TK2 in mitochondria. Although TK sources of body fluids and the mechanism is unclear, TK protein itself does not have the signal peptide, but speculated fluids of TK is likely to be the cell disintegration or secreted by tumor cells, therefore, TK vigorous growth as an indicator can be used as cells. View TK rise or reduce the occurrence of tumor development, metastasis of [1,2]. The research group [3 6] using recombinant DNA technology, cloning the TK gene, expressed in E. coli TK by immunization of Balb / C mice to produce monoclonal antibodies and established a method for the quality of the TK detection enlarged pressure electric immune sensor. The this experiment quality amplification the piezoelectric immunosensor were detected in the serum of healthy individuals, benign tumors, and liver cancer, pancreatic cancer, stomach cancer and other malignancies TK to investigate the quality amplification the piezoelectric immunosensor detected by serum TK and as the feasibility of a new marker in malignant tumors, to lay the foundation for further TK detection for malignancy clinical diagnosis.
1 Materials and Methods
1.1 Selection of cases
Experimental specimens were from Zhongshan Hospital of Hubei Province and Hubei Cancer Hospital cancer patients. Including 69 cases of liver cancer, male 61 cases, 8 females, age 23 to 80 years old, the average age (53.65 ± 12.25) years old; gastrointestinal cancer, 39 patients, 22 males and 17 females, aged 28 to 81 years, with an average (61.89 ± 15.33) years of age; 25 cases of lung cancer, 17 males and 8 females, aged 36 to 75 years old, the average age (57.33 ± 11.25) years old; reproductive system cancers in 26 cases, 6 males and 20 females, aged 36 to 75, ages 25 to 82 years old, the average age (55.52 ± 13.61) years old; 13 cases of breast cancer and other malignancies, are women, age 37 to 64 years old, the average age (49.70 ± 8.05) years old; 21 cases (5 cases of bile duct cancer, pancreatic cancer, kidney cancer patients, brain metastases, nasopharyngeal three cases of pancreatic cancer one cases, one cases of malignant pleural effusion, lung metastasis of gastric cancer patients, submandibular gland carcinoma adenocarcinoma lymph node metastasis in 1 case), 11 males and 10 females, aged 41 to 83 years old, the average age (57.25 ± 17.57) years; benign tumors in patients with 15 cases, 4 males and 11 females, aged 27 to 70 years, mean age (47.60 ± 10.13) years; healthy human serum 30 cases, 13 males and 17 females, all serum samples were hospital routine morning venous blood collected separation, collected and stored at -20 ℃.
1.2 Instruments and reagents
The CN3165-type high-resolution counter (Shijiazhuang Wireless Electrical Appliance Factory); quartz crystal: AT cutting (JA5 type), 9 MHz, both sides of the gold electrode chip diameter 12mm, electrode diameter 6mm oscillation circuit laboratory homemade TTL circuits. TK, anti-TK monoclonal antibody the (Anti TKAb) and horseradish peroxidase enzyme-labeled anti-TK monoclonal antibody the (HRP Anti TKAb) are the preparation of the study group, the method has another paper reported [3 5].
The quality of the research group reported amplification Piezoelectric Immunosensor method , prepared by the research group Anti TK Ab sulfhydryl self-assembled monolayer covalently fixed on a piezoelectric quartz crystal surface, the prepared hTK piezoelectric immunosensor The serum samples diluted 1:1 was measured.
1.4 statistical methods
T test were calculated of different malignancies serum and normal human serum, of malignancy serum and benign serum as well as benign serum with normal serum of the frequency shift between the value of the statistic t analyze the meantime differences.
The 2.1 TK serological detection standard curve
TK solution of a series of different concentrations were measured (pure product formulated with a TK) to obtain the the TK concentration and the sensor frequency response curve, Figure 1. Obtained by linear regression analysis, the frequency shift values of TK concentration linear correlation equation Y = 77.4-14.7X, and γ = 0.9955.
Figure 1 sensor frequency response of the concentration of the standard curve with TK
2.2 TK serological testing
Adopt quality zoom the piezoelectric immunosensor detection of different malignancies serum, the benign serum and normal human serum, are shown in Table 1. The results show that the liver cancer serum TK highest level, the average of the 69 cases of liver cancer serum frequency shift value (59.4 ± 3.38) Hz TK levels (2.44 ± 0.46) ng / ml; followed by breast serum, the frequency shift value (68.9 ± 1.75) Hz, TK level (1.16 ± 0.24) ng / ml. Both TK level there are significant differences. Table 1 clinical samples TK serological test results a: control; b: the benign serum control; c: serum and breast cancer control; d: Serum controls with other malignancies; – with normal serum: concentrations of less than linear range at the same time, both the TK water was significantly higher than that of other malignancies serum (P <0.001). 0.05）。">Lung cancer, gastrointestinal cancer, reproductive system cancer and other malignancies serum TK level approaches, the difference was not statistically significant (P> 0.05). Benign tumors in patients with normal serum TK level lower, below the sensor linear detection range of all malignant tumors in the experimental serum sample frequency shift value of their difference was statistically significant (P <0.001). In addition, the frequency shift of the benign tumor serum also significantly greater than normal serum TK level difference was statistically significant (P <0.001).
The TK energy conversion between the reversible catalytic thymine nucleoside and phosphate nucleoside, is closely related to cell division and proliferation, is a more sensitive tumor markers [1,2]. International still mainly by radioimmunoassay determination of serum TK activity and content, but the long-term radioactive method, there is a certain amount of radioactive contamination, and requires expensive equipment and radioactive reagents, it is difficult to promote the use of clinical diagnosis and screening.
The study group independently prepared by the TK and corresponding anti-TK monoclonal antibody, and anti-TK monoclonal antibody immobilized on the piezoelectric quartz crystal surface, the developed quality zoom TK piezoelectric immunosensor. This sensor with high sensitivity, easy to operate, easy to automate, digitize, without the need for expensive equipment, use less reagent use, inexpensive, and linear regression analysis showed that the frequency shift of the sensor response with the TK concentration has very A good correlation (γ = 0.9955), and can be accurately obtained Tk concentration information. On this basis, the present experiment measured TK levels of a number of different tumor types of serum samples, the results show that whether it is malignant or benign serum frequency response than that of healthy people sera, indicating that cancer patients due to the excessive proliferation of cells , TK as a key enzyme for DNA synthesis and cell division is closely related to the expression of TK protein rapidly increase serum TK level also significantly increased, and the difference was statistically significant with healthy human serum. At the same time, we still found that the serum samples of different types of cancer, its TK level, there is an apparent difference, hepatoma serum highest (2.44 ± 0.46) ng / ml, followed by breast cancer serum (1.16 ± 0.24) ng / ml, two The TK level for the difference was statistically significant, and are significantly higher than other malignancies serum (P <0.001). 0.05）。">Lung cancer, gastrointestinal cancer, reproductive system cancer and other malignancies serum TK level close to, the difference was not statistically significant (P> 0.05). Visible TK as a general indicator of cancer screening, and can be easily distinguished qualitatively liver cancer, breast cancer and other malignant and benign tumors, therefore, the quality of the experimental application amplification TK the piezoelectric immune sensor method as a simple, sensitive TK New measurement methods used in clinical tumor census, is expected to promote and distinguish tumor types, especially in the aspect of the clinical diagnosis of liver cancer, breast cancer has a good prospect.