Responsive Photonic Crystal Based Suspension Array In The Application Of Tumor Diagnosis And Treatment

Cancer is one of the leading causes of human death because of its high morbidity and mortality.China is the most populous and largest developing country and its population has become increasingly aged,which make tumor burden more prominent and bring huge economic or social pressure to China.Therefore,the prevention,diagnosis and treatment of cancer are important issues to be solved by the Chinese public health service.Secondary prevention is the key cancer prevention measure,namely early detection,early diagnosis and early treatment,which requires effective tumor detection or monitoring methods.These methods screening,diagnose or monitor cancer mainly by pathological and imagining examination as well as body liquid composition examination.In which,pathological examination is usually regard as gold standard,but its disadvantages such as invasive process,high technical requirements for personnel and time-consuming limit its application.Imaging method still requires special equipment that may expose personnel to radiation and may be expensive.In contrast,body liquid composition examination has been widely used in screening,diagnosis and monitoring of tumor owning to many advantages such as being source abundant and available,convenient,quick,noninvasive,sample batch processing,and dynamic service system to monitor.However,the traditional detection methods of body liquid composition have some disadvantages including low efficiency,insufficient specificity or sensitivity,therefore,their application is greatly limited.One of the main ways to improve detection efficiency is to adopt multiplex and high-throughput detection,such as suspension array;Choosing tumor markers with high specificity can greatly enhance the detection credibility,such as detection of circulating tumor cells(CTCs),which directly and accurately reflects their sourced tumor sites and is regarded as a kind of liquid biopsy of tumors.Photonic crystals are three-dimensional ordered nanostructured materials with properties of characteristic stable reflection peak and large surface-to-volume ratio(SVR).Their characteristic reflection peak of photonic crystal can provide a stable encoding for suspension array,which can avoid the problem of quenching or interference in fluorescence encoding.Their high SVR can greatly enhance the sensitivity of suspension array.However,photonic crystals based on common forms such as silica colloidal crystal beads(SCCBs)still have many problems,such as high background values,poor biocompatibility.Therefore,the suspension arrays based on them have limited sensitivity and they are not suitable enough to be carriers of CTCs,which restrict their application in secondary prevention of the tumor.Based on the aforementioned background,this dissertation developed new types of photonic crystals,which are responsive and customized to multiplex screening of tumor markers,detection of CTCs and multiplex analysis of multi-drug resistance genes,respectively.My works are mainly divided into three sections and their respective innovation points are presented subsequently:1.The application of suspension array technology based on response inverse opal photonic crystal in multiplex detection of tumor markers.In this section,two types of inverse opal photonic crystal microbeads were fabricated to form suspension arrays for multiplex detection of tumor markers.The microbeads were mainly generated by poly(ethylene glycol)diacrylate(PEGDA)gel and poly(N-isopropylacrylamide)(pNIPAM)gel respectively,which was responsive to stimuli,solvent and temperature respectively.At the same time,a microfluidics chip was developed to combine with the solvent suspension array for its high repeatability and better controllability.Before signal measurement,they were stimulated by ethyl alcohol or higher temperature to shrink their volume and increase the density of fluorescent molecules,thus realizing signal self amplification and increasing sensitivity.The suspension arrays based on them had advantages of low fluorescence background,low non-specific adsorption,large available area and signal self-amplification.Using standard solution and clinical specimens,the suspension arrays were shown to be quick,accurate and sensitive enough in simultaneously detecting two tumor markers,alpha-fetoprotein(AFP)and carcinoembryonic antigen(CEA),suggesting they were suited to cancer screening of large populations.2.Folic acid-functionalized hybrid inverse opal barcode for capture and release of circulating tumor cellsCTCs are tumor markers with high specificity,which can provide more direct and precise tumor information.However,it is a tremendously challenge to separate and measure them for their extremely low concentration.The current methods are based on their physical properties or capture molecules such as epithelial cell adhesion molecule(EpCAM)to separate them,which are nonspecific,complex,expensive and easily losing efficacy.Folate receptors(FRs)are commonly overexpressed in cancer cells while expressed at low to negligible levels in most normal tissues and they are targeting ligands of folic acid(FA)with high binding affinity.Therefore,the use of FA-FR interaction has potential application in CTCs detection.For utilization of CTCs detection by FA-FR interaction,a suitable carrier is needed.Photonic crystal is a potential candidate owning to its nano-structure and large SVR.While their biocompatibility and stability cannot be satisfied at the same time for common photonic crystals.In this section,we developed a new type of hybrid inverse opal photonic crystal accustomed for capture and release of CTCs by FA-FR interaction.The hybrid inverse opal barcode was composed of two nano-orderd building blocks,PEGDA and methacrylated gelatin(GelMA)gel.PEGDA gel with their good mechanical strength and GelMA with their abundant amino group could provide sufficient binding sites for FA coupling and their soft texture could decrease impairment to CTCs during the CTCs-barcode contact.Ferroferric oxide magnetic nanoparticles(FOMNPs)endowed the barcodes with response to magnetic field,which could greatly facilitate separating process after cell capture.It was demonstrated by cell model or blood tumor model that FR positive CTCs could be captured accurately with good sensitivity,reliability and specificity,and the captured cells could be released with good viability.These features make the FA-functionalized hybrid inverse opal barcode promising for capturing,detecting,and releasing CTCs,which could be helpful to early detection,diagnosis and treatment of cancer.3.The application of suspension array technology based on response inverse opal photonic crystal in multiplex analysis of multi-drug resistance genes.Early treatment is another key to secondary prevention of cancers,which relies on drugs greatly.However,drug treatment will inevitably encounter a problem,namely drug resistance.By analyzing cell gene or protein,the multi-drug resistance(MDR)expression profiling could be obtained and then personal therapeutic strategies could be prepared for preliminary treatment and reversal of MDR,which not only increases curative effect,but also decreases side effect.MDR of tumor cells could be caused by many mechanisms,of which ATP binding cassette(ABC)membrane transport proteins,such as P-glycoprotein(P-gp)and multidrug resistance-associated protein 1(MRP1),are the most important.The expression level of their encoding genes,MDR1 and MRP1 gene can be obtained early because their gene expression usually precedes gene amplification,which is important for early MDR detection.While the existing gene detection methods such as quantitative PCR are either insensitive or incompetent for multiplex detection,which are not able to comprehensively profile MDR gene expression early because these MDR genes work separately or simultaneously and their type even subtypes are various.In this section,suspension arrays based on two types of response inverse opal photonic crystals were developed for multiplex analysis of MDR genes.One was pNIPAM gel based responsive inverse opal photonic crystal,the other was polyacrylamide(PAM)gel based.Similar to section 1,the response properties of the two microbeads were utilized to enhance signal intensity to get higher sensitivity for multiplex analysis of MDR genes.The feasibility of this project is preliminarily explored by standard solution.In conclusion,the responsive photonic crystals with their unique nanostructures,good biocompatibility,stimuli responsive property and stable code capacity have a good application prospect in suspension array.This dissertation comprehensively explores their application in multiplex detection of tumor markers,CTCs detection and multiplex analysis of MDR genes,which showed promising roles in secondary prevention.This dissertation has certain practical application values and is worthy of further research and development.