Construction of Surface-enhanced Raman Spectroscopy Biosensor Based on Nanomaterials and Nucleic Acid Signal Amplification Strategy

Author:He Yi

Supervisor:chai ya qin yuan ruo


Degree Year:2019





Surface-enhanced Raman spectroscopy(SERS),with the advantages of rapid,sensitive,nondestructive analysis and fewer samples required,has been widely applied in the fields of marked and unmarked detection,material science,biochemistry,biomedicine,electrochemistry and molecular imaging and so on.Surface-enhanced Raman spectroscopy biosensor has also become an important tool for detection of disease markers and single-molecule analysis.When construction of surface-enhanced Raman spectrum biosensor,some new nanomaterials were often used as SERS substrates with strong enhancement effect to improve the sensitivity of the sensor,and it also combined with efficient signal amplification strategy to further improve the performance of surface-enhanced Raman spectrum biosensor.The development and construction of these new materials and methods are of great significance for sensitive and specific detection of various biological molecules,and it can also provide new ideas for the practical application of SERS biosensors.In this paper,a variety of signal amplification strategies were designed to construct novel SERS biosensor.By combining new nanomaterials and various nucleic acid signal amplification strategies,SERS biosensors with high sensitivity,simple operation and high stability were prepared for sensitive and rapid detection of analytes.The details are mainly as follows:1.Research on sandwich immune surface enhanced Raman spectroscopy biosensor based on metal organic framework materials and magnetic cobalt ferrite nanoparticleDue to its good specificity and high sensitivity,SERS biosensors are often used in the sensitive detection of disease markers.In order to improve the sensitivity of the sensor,this study adopted a new metal organic framework particle combined with tetrapod Au nanoparticles to as SERS substrate(IRMOF-3@AuTPs)for sensitive detection of NT-proBNP.Firstly,the SERS substrate IRMOF-3@AuTPs was supported with Raman signal molecule toluidine blue(TB)and primary antibody(Ab1).Due to the large specific surface area and good biocompatibility of IRMOF-3,a large number of primary antibodies and TB can be supported.And due to the"hot spots"generated by AuTPs,the Raman signal of TB can be significantly enhanced.Then the second antibody was immobilized on CoFe2O4.With the presence of NT-proBNP,for the specific binding capacity between antigen and antibody,the SERS signal part of the second antibody was immobilized on the base,thus achievingdetection range from 1 fg mL-1 to 1 ng mL-1 with a detection limit of 0.75 fg mL-1.And this newly designed amplification strategy holds high probability for ultrasensitive immunoassay of NT-proBNP.2.An ultrasensitive SERS biosensor based on rolling circle amplification for sensitive detection of miRNAAt present,it is far from enough to use new nanomaterials to improve SERS subsrate for improving the sensitivity of the SERS sensor.As a result,some new amplification technologies needed to be explored to consturct SERS biosensor for realizing sensitive detection of cancer markers.In this study,the target induced rolling ring amplification technology was adopted into SERS biosensor.And a new nanomaterials Co/C@Ag were conmbine with SERS for construction of SERS biosensor.,which not only had large specific surface area and good biocompatibility,but also had good magnetism,to build SERS sensor.First,target miRNA can induce rolling ring amplification to generate a large number of primer DNA,and this cycle can achieve exponential amplification.Secondly,Co/C@Ag as SERS substrate can significantly enhance the peak of Raman signal molecule,thus amplifying the sensitivity of biosensor.When the obtained primer chain DNA was added to the Co/C@Ag substrate,the Raman peak of the substrate would be significantly changed due to the occurrence of chain replacement.Based on such a principle,the purpose of sensitive detection of miRNA was realized.This SERS strategy exhibited a wide linear range of 100 amol L-1 to 100 pmol L-1 with a low detection limit of 70.2 amol L-1,which indicated the proposed SERS platform has potential application value for ultrasensitive bioassay of miRNA.3.The construction of ultrasensitive SERS biosensor based on 3D DNA hydrogel amplification and ficus virens leaves as SERS substratesIn the construction of SERS biosensor,it was often required to label Raman signal molecules on the substrate,but the process of marking was often complex and the number of markers was limited,which also affected the sensitivity of the sensor.In this study,a novel three-dimensional DNA hydrogel structure was used for construction of SERS biosensor.The Raman signal molecule toluidine blue(TB)was encapsulated in the DNA hydrogel by ingenious design of DNA chain.Meanwhile,the complementary pairing principle of DNA bases was used to regulate the hydrogel structure and release the signal molecule to achieve the purpose of detecting the target.First,during the preparation of DNA hydrogel,a large amount of TB was wrapped into the structure of DNA hydrogel.At this point,the target miRNA can trigger a DSN-induced cyclic amplification to obtain a large number of released DNA chains,which can open the DNA hydrogel to release TB.At the same time,this study used the ubiquitous Ficus virens leaves combined with Ag nanoparticles as SERS substrates,which was economical,environmentally friendly,and had good enhancement effect ands stability.Since silver nanoparticle had a good enhancement effect on Raman signal molecules,when TB was released to SERS substrate,a strong Raman signal was generated to realize the detection of target miRNA.As a result,the as-proposed SERS platform can sensitively and selectively detect miRNA 155 with a wide linear range of 0.1 fmol L-1 to100 pmol L-1 and low detection limit of 0.083 fmol L-1,which indicated that the biosensor has great potential to be applied in miRNA-related clinical diagnostics and biochemical researches.4.The construction of ratiometric SERS biosensor based on DNA hydrogel amplificationWhen SERS biosensors were used to detect biological samples,the accuracy was often doubtful.And the traditional SERS sensors often used two kinds of signal molecules to design ratio sensors to improve the accuracy and credibility of SERS analysis.However,there are often drawbacks to using two probes,such as complex labeling process and expensive chemicals.In this study,we proposed only labeling a Raman probe,3-mercapto phenylboric acid(3-MPBA),to construct SERS sensor for sensitive detection of target.The method has simple operation procedure and low cost.The 3-MPBA has a unique Raman peak at 996 cm-1 can be a standard reference material.We fixed it to the silicon wafer@Nafion(Si@Nafion)SERS substrate.When the DNA hydrogel coated with glucose oxidase(GOx)is opened by the released DNA obtained through the cyclic amplification process,GOx can be released from the DNA hydrogel.So glucose oxidase can catalyze glucose to produce hydrogen peroxide on Si@Nafion.Subsequently,the obtained hydrogen peroxide can further react with 3-MPBA to produce 3-hydroxyphenylthiophenol to produce a new Raman peak at 883 cm-1.In this process,the peak strength of 996 cm-1 remains unchanged,so 996 cm-1 can be used as a reference standard.This method greatly improves the reliability of the sensor for object detection.As a consequence,our SERS biosensor can sensitively detect miRNA 122from 10 amol L-1 to 100 pmol L-1 and the detection limit was 7.75 amol L-1.Our strategy adopts a novel ratiometric method with one Raman probe to detect miRNA,opening a new avenue for trace amount of biological sample detection with high sensitivity and accuracy.5.The construction of SERS biosensor based on a dual signal amplification strategyIn the construction of SERS biosensor based on nucleic acid amplification strategy,some drawbacks such as the wasting of nanomaterials and DNA chain,which also hindered the improvement of sensitivity of biosensor.If the prepared materials and DNA chains can be used reasonably,the sensitivity of biosensor will be further improved.In this study,the inclusion reaction induced by the target miRNA was introduced to aggregate the ZnO@S1/S2 and CoFe2O4@S3 complexes.When hydrochloric acid is added,a large amount of ZnO can be dissolved to obtain a large amount of Zn2+,which can further induce DNA enzyme amplification cycle.At the same time,through ingenious design of DNA,complementary DNA strand with DNA enzyme can be cut into two DNA strand with similar sequence for the next sensing research,which greatly improves the utilization rate of DNA strand and amplifies the signal at the same time.Therefore,the conversion of nanomaterials into metal ions can achieve a great enhancement effect,and the effective use of DNA chain further amplifies the signal of the sensor.As a result,this double amplification strategy based SERS biosensor can achieve a low detection limit of 6.82 amol L-1 and wide linear range from 10 amol L-1 to 10 pmol L-1,which shown great potential in the clinical application and medical diagnosis..