The Development Of Novel Optical Biosensor Strategies And Their Application In Tumor Marker Detection

Malignant tumor is a kind of high risk disease which is a great threat to human life,its morbidity and death number are increasing year by year.The detection of tumor markers has important value for the early diagnosis,therapy evaluating and prognosis judgment of tumors,but the current clinical detection methods still have some limitations in the analysis of tumor markers.Biosensor is a device that uses bioactive substances as identification elements to output signals from the identified targets through conversion elements to achieve detection purposes.It has the advantages of high sensitivity,high specificity and simplicity,among which optical biosensor is one of the most widely used one.Isothermal amplification technology is a controllable nucleic acid amplification technology,which has the advantages of high efficiency,simplicity and rapidity,and can make up for the defects of existing nucleic acid amplification methods.Nanomaterials provide a new tool for biosensors developing for their unique optical,stability,magnetic,electrical and other characteristics.Combining the developed the lack of biosensor and the actual development needs of clinical laboratory diagnostics,this thesis mainly are based on isothermal amplification and novel nanomaterial to build a series of high efficiency,simple optical biological new strategies to realise detections of point mutations,circulating tumor cells and specific nucleic acid sequence of high sensitivity with high specificity and sensitivity,supporting the detecting for clinical disease markers and the reserch of biosensors.The main research contents are as follows:1.High-sensitive colorimetric biosensing of PIK3 CA gene mutation based on mismatched ligation-triggered cascade strand displacement amplificationThe development of highly sensitive assay for low abundance cancerrelated gene mutation is significant for early diagnosis and personalized medicine of cancer.We developed a novel colorimetric strategy to determinate PIK3CA^H1047R gene mutation sensitively and specificly by combining mismatched ligation-triggered cascade strand displacement amplification（SDA）with G-quadruplex/hemin DNAzyme-catalyzed colorimetric biosensing in this work.Meanwhile,mismatched bases and mutation complementary location of ligation probes were optimized detailedly to obtain superior capacity of PIK3CA^H1047R gene mutation determination.The mismatched ligation system selectively triggered downstream cascade SDA to produce a great abundance of G-quadruplex sequences.Subsequently,the numerous G-quadruplexes could combine with hemin to form G-quadruplex/hemin DNAzymes,catalyzing a colorimetric reaction for mutation detection.Based on this mismatched ligation-triggered cascade SDA,the developed method shows admirable capability for mutation determination and high-efficiency for signal amplification.The designed biosensing strategy could detect as low as 0.2% PIK3CA^H1047R mutation.In addition,this biosensing strategy could be scalable for the analysis of low abundance mutation gene spiked into human serum samples.Thus,this colorimetric biosensor might become a potential alternative tool for genetic analysis and clinical molecular diagnostics.2.A novel sandwich-like cytosensor based on aptamers-modified magnetic beads and carbon dots/cobalt oxyhydroxide nanosheets for circulating tumor cells detectionAs a part of "tumor fluid biopsy",circulating tumor cells（CTCs）contain enormous tumor information,so the detection of CTCs is very helpful in clinical diagnosis and therapy guide.Herein,we applied aptamers-modified magnetic beads（Apt@MBs）and carbon dots/cobalt oxyhydroxide nanosheets systems（CDs/CoOOH）to develop a new cytosensor for ultrasensitive and specific detection of CTCs.At first,amino-modified magnetic beads（MBs）were cross-linked with carboxylmodified MUC1 aptamers（Apt）to gain Apt@MBs.Then,folic acid（FA）functionalized carbon dots（FA@CDs）were assembled onto cobalt oxyhydroxide nanosheets（CoOOH）to obtain FA@CDs/CoOOH with fluorescence quenching.In the detection systems,Apt@MBs identified and accumulated MCF-7 cells,forming Apt@MBs/MCF-7 cells,which then combined with FA@CDs/CoOOH to obtain sandwich-like Apt@MBs/MCF-7 cells/FA@CDs/CoOOH.Finally,ascorbic acid（AA）decomposed CoOOH to release carbon dots（CDs）,and the fluorescence of abundant CDs act as efficient signal output.The cytosensor detected MCF-7 cells with a wide dynamic range（10 10⁵ cells/m L）and a low limit of detection（5 cells/m L）,owing to the specific identification and effective enrichment of Apt@MBs as well as anti-interference capability and signal amplification of FA@CDs/CoOOH.Most importantly,the cytosensor successfully discriminated MCF-7 cells from white blood cells,exhibiting potentiality in ultrasensitive CTCs detection in clinical diagnosis.3.A novel biosensor for PML/RARα fusion gene detection based on dual signal amplification strategy of isothermal amplification and cobalt oxyhydroxide nanosheets/quantum dotsA novel biosensor was developed by combing triple-stranded DNA hybridization-triggered strand displacement amplification（tri-HT SDA）and cobalt oxyhydroxide nanosheets/quantum dots（CoOOH/QD）-based amplification to detect PML/RARα fusion gene（P/R）.We first used both isothermal amplification and nanomaterial amplification for specific DNA sequence detection.Firstly,QD was assembled on CoOOH to obtain CoOOH/QD,and then modified with PEI,which was linked onto Fe₃O₄/Au by ligation probe（LP）,forming Fe₃O₄/Au@LP@PEI/CoOOH/QD.In the detection system,detection probes could discriminate P/R to form triplestranded DNA hybridizations and trigger downstream SDA.Then,the accumulated tri-HT SDA products（DNAzymes）were obtained by continuously strand displacement extension and cleavage.Afterwards,the obtained DNAzymes would bond with and cleave LP,releasing CoOOH/QD into the supernatant.Finally,ascorbic acid decomposed CoOOH to releases QD,resulting an effective signal output.Owing to the specific recognition and efficient amplification of tri-HT SDA as well as impressive anti-interference and considerable amplification of CoOOH/QD,this biosensor showed a wide dynamic range（10 fM 10 nM）and a low limit of detection（5.50 fM）in P/R detection,showing potentiality in sensitive detection of specific DNA sequence in clinical diagnosis.