Establishment Of Nucleic Acid Tumor Marker Detection Platforms Based On ID Ring/3D Coded Microspheres And THz And Their Applications In Colorectal Cancer

BackgroundColorectal cancer is one of the most common malignant tumors of the digestive system.Early screening and diagnosis can improve the five-year survival rate of cancer patients.However,the early lesions of colorectal cancer are mostly located in the mucosal layer and submucosa,thus it lacks the typical early clinical manifestations.Traditional screening methods include colorectal endoscopy and fecal occult blood test,but colonoscopy is an invasive test,and fecal occult blood screening test has poor specificity.Serological tumor markers are used to screen suspected patients and high-risk groups.It has become the most ideal clinical early non-invasive screening method.Although the existing tumor markers（CA199,CEA,CA242,etc.）have a certain increase in the early stage of the tumor,the sensitivity and specificity of a single tumor marker are not high.The combined detection and analysis of multiple tumor markers and existing clinical test indicators can make up for the shortcomings of a single tumor marker to a certain extent,but a more convenient,accurate and efficient method is still needed to improve the diagnostic efficiency of tumors.Tumor diagnosis and treatment have entered the era of precision medicine.In the context of precision medicine,liquid biopsy,as a convenient,minimally invasive,and dynamically reflecting tumor gene profile,is playing an increasingly important role in clinical research and practice.Compared with protein tumor markers,nucleic acid tumor markers can reflect the pathophysiological changes of the body from the molecular level earlier.In addition,nucleic acid tumor markers can be paired with specific bases of target m RNA to regulate gene expression in a specific manner,so they can be used as targeted therapy tools.Therefore,the construction of integrated diagnosis and treatment technology based on nucleic acid tumor markers has become one of the most valuable methods for accurate diagnosis and treatment of colorectal cancer.However,the existing nucleic acid tumor marker detection technologies such as amplification refractory mutation system（ARMS）,digital PCR（d PCR）,next-generation sequencing（NGS）,reverse transcription-polymerase chain reaction（RT-PCR）usually requires professional operations,complicated primer design,and multiple temperature cycles,which limit the further clinical application of nucleic acid tumor markers.In summary,this study has established new biosensing platforms for different colorectal cancer-related nucleic acid tumor markers including circulating tumor DNA（ct DNA）and micro RNA（mi RNA）,and their clinical efficacy.The evaluation has provided new methods and ideas for the application of liquid biopsy in the early diagnosis of colorectal cancer.In addition,the relationship between the coagulation function indexes,tumor markers and clinical stages of colorectal cancer patients and non-colorectal cancer patients is explored,which can provide a certain reference value for the early diagnosis of colorectal cancer.Objectives1.To explore the relationship between coagulation function indexes and tumor markers and clinicopathological characteristics in patients with colorectal cancer;2.Establish a ct DNA multiplex detection platform based on the 3D coded ID rings and evaluate its performance in the detection of colorectal cancer clinical samples;3.Establish a mi RNA-21 terahertz metamaterial sensing platform based on duplex-specific nuclease-triggered rolling circle amplification.Methods1.Collect blood coagulation function indexes and tumor markers of patients with colorectal cancer,patients with benign lesions of colorectal cancer and healthy people,and carry out the statistical analysis.2.Interlocked DNA ring（ID ring）identification unit is designed,containing a recognition ring（for ct DNA recognition）and a reporter ring（for signal report）.The recognition ring has a sequence complementary to ct DNA.When the recognition loop and ct DNA recognition complement each other to form a DNA double strand,specific restriction endonuclease（RE）can recognize the mutation site（single base mutation）in ct DNA and cut it to open the recognition ring and release the reporter ring.The reporter ring can start rolling circle amplification（RCA）,so it can realize the specific and sensitive detection of ct DNA.The ID ring identification unit was characterized and optimized by polyacrylamide gel electrophoresis（PAGE）,atomic force microscopy,etc.Hemin-G-quadruplex DNA enzyme catalyzing substrate 3,3’,5,5’-Tetramethylbenzidine（TMB）color experiment and fluorescence signal detection were used for result outputs.3.A three-dimensional（3D）coded unit is designed in the suspension microarray.In the3D coded unit,two different fluorescent dyes are used to code the microspheres.The combination of the two fluorescent dyes can be used as the 2D codes（X,Y）of different targets ct DNA by adjusting the ratio of the two fluorescent dyes.When the target ct DNA is present,the fluorescent protein signal（fluorescence intensity）bound by the amplified RCA product is used as the Z coordinate.Based on this,（X,Y）is established as the 2D codes for determining the target type,and the Z signal is used as the（X,Y,Z）3D coded unit for determining the amount of ct DNA.4.By integrating the above ID ring identification unit and 3D coded unit,a multiplexed ct DNA detection platform based on the 3D coded ID rings is established.Four kinds of ct DNAs with KRAS,NRAS,PIK3CA and BRAF mutations of colorectal cancer were selected as targets.Choose four 2D codes as X₁Y₁,X₁Y₂,X₂Y₁,and X₂Y₂for the codes of the four target ct DNAs respectively,and use phycoerythrin to combine with the obtained RCA products,and the signal intensity of phycoerythrin represents the third-dimensional signal Z,thus establishing the 3D codes of different targets.A mixture of negative control samples and four ct DNA samples were used as a control to verify the specificity of the platform,and synthetic ct DNAs with different concentrations were used as quality control to verify the sensitivity of the product verification platform.We collected clinical samples（blood,feces,urine）to determine the mutation types of four target ct DNAs through the established platform and traditional sequencing methods,and compared the consistency of the two methods through chi-square test.5.A DNA capture probe was designed with mi RNA-21 as the target,and coupled to the magnetic beads.When the target is present,the probe is captured to form a DNA-RNA complex,and double-stranded specific nuclease（DSN）can recognize the complex,degrade the DNA part and expose the primer part on the DNA probe.After the primer part is exposed,RCA is amplified in situ on the surface of the magnetic beads.By using the terahertz metamaterials to measure the signal difference of the magnetic beads before and after the amplification to obtain the signal value of the nucleic acid amplification products,the mi RNA-21 sensing platform based on the terahertz metamaterial technology is established to realize the highly specific and sensitive detection of the target.Results1.The high level of fibrinogen（Fib）is possibly related to the deep infiltration of the primary tumor and regional lymph node metastasis;tumor markers may further increase in the case of regional lymph node metastasis and distant metastasis of colorectal cancer;Fib,D-Dimer（D-Di）and CA242 in early stage（stage I）of cancer patients are significantly higher than those with benign lesions.2.Atomic force microscopy results suggest the formation of a DNA interlocking double ring structure.PAGE results showed that in the presence of the target ct DNA,the interlocked double-ring structure of DNA can be cleaved by RE and then trigger RCA to produce RCA products.Through optimized screening,base numbers of the optimal ID rings for four targets of KRAS,NRAS,PIK3CA and BRAF are 28 bp,24 bp,28 bp and 32 bp,respectively.The results show that the detection limit of the TMB colorimetric test for KRAS,NRAS,PIK3CA and BRAF can reach 0.4%,0.3%,0.4%,0.5%respectively.Then,the detection limits of the fluorescent test for KRAS,NRAS,PIK3CA and BRAF can be further increased to 0.3%,0.1%,0.2%,0.4%.3.The successful establishment of the ct DNA multiplex detection platform based on the3D coded ID rings is divided into two aspects for verification.In terms of specificity,the platform can distinguish between negative control samples,four positive control samples（containing only one target ct DNA）,and mixed samples（containing four target ct DNA）;in terms of sensitivity,the platform can distinguish between KRAS,NRAS,PIK3CA and The detection limits of BRAF can reach 0.05%,0.1%,0.1%,and 0.05%respectively,which are significantly better than traditional sequencing methods.The detection and statistical analysis of 187 blood samples,20 stool samples and 20 urine samples of colorectal cancer patients with our multiple detection platform and traditional sequencing methods respectively show that the detection results of the two methods have good consistency（P>0.05）.4.The feasibility of DSN is successfully verified triggering the RCA system through PAGE,and then successfully established a biosensing platform based on terahertz metamaterial technology.Under the optimal conditions,the linear range of the sensor is 10 f M to 10 n M,and the detection limit is 8.49 f M.It has the ability to identify other interfering mi RNAs and good repeatability.Conclusions1.There is a certain positive correlation between tumor markers and coagulation function indicators in patients with colorectal cancer.Although the diagnostic criteria are not yet formed,it can be used as a powerful supplement and supporting evidence for early screening and prognostic judgment in addition to clinical manifestations and pathological biopsy.2.A ct DNA multiplex detection platform has been successfully established based on the3D-encoded ID rings.The platform has good consistency with the sequencing method in terms of clinical sample detection performance,but our platform takes shorter time and lower cost and has a higher detection sensitivity（up to 0.05%）.3.Based on the terahertz metamaterial technology,a biosensor combined with DSN triggered RCA has been established to achieve highly sensitive and specific detection of target mi RNA.