Establishment And Preliminary Application Of New Technologies In Clinical Laboratory Diagnostics Based On Graphene Oxide

Objectives: There are many shortcomings in the current methods for detection of genitourinary mycoplasma and determination of C-peptide in clinical laboratory.Graphene oxide exhibits unique 2-D structural,chemical,and electronic properties that lead to its many potential applications.In order to expand the scope of its usage,two clinical detection platforms for DNA detection in genitourinary mycoplasma and C-peptide determination in diabetes serum based on graphene oxide were developed.I.Establishment of LAMP-GO-LAPS(Loop-mediated isothermal amplification combined with graphene oxide modified light addressable potentiometric sensor)assay for the detection of genitourinary mycoplasmaMethods:1.Graphene oxide was prepared by improved Hummers assay.2.After first pass by the inclusion and exclusion criteria for primer design,primer screening of UU,MH and MG were performed by gel electrophoresis and turbidimeter.3.Sensitivity and specificity of the LAMP assay were explored using optimal primers.Reaction time and temperature were optimizatized.4.A simple boiling method for DNA extraction as clinical sample preparation was tested.5.Probes of LAMP-GO-LAPS chip for detection of urogenital mycoplasma were designed and screened.6.Hybridization conditions of LAMP-GO-LAPS chip for detection of genitourinary mycoplasma were improved.7.Comparison of LAMP-GO-LAPS assay with PCR assay for detection of genitourinary mycoplasma was completed in clinical samples.Results:1.Improved Hummers method was used to prepare graphite oxide.2.The best primers for UU,MH and MG were successfully screened out,separately.3.LAMP assay was developed for rapid amplification of three genital mycoplasmas,and using a simple boiling method for DNA extraction.4.LAMP-GO-LAPS detection platform was successfully established and applied for detection of genitourinary mycoplasma in clinical samples.5.Multiplex LAMP-GO-LAPS assays for detection of three genital mycoplasmas was initially developed.II.Rapid quantification of C-peptide in diabetes patients by immunoaffinity enrichment and MALDI TOF Mass SpectrometryMethods:1.Several potential nanoparticle enrichment platforms for their ability to act as MALDI comatrixes that enhance the MS signal by increasing C-peptide desorption/ionization were analyzed.2.C-peptide was enriched from serum samples using magnetic beads.Unrelated proteins were partially depleted in the process.3.The enriched C-peptide was modified with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate(AQC).4.After Ziptip,the samples were ready for analysis by MALDI TOF MS.5.Graphene oxide was used as comatrix for determination of C-peptide.6.The recombinant human C-peptide compared against the reference standard C-peptide from NIH was used as a calibrator and a stable isotopic-labeled human C-peptide was used as internal standard.7.Method validation for the new assay was performed.Results:1.A new standardizes C-peptide measurement assay has been developed and validated according to guidelines with a limit of quantitation of 25 p M.2.A correlation between the GO-MS assay and the reference method was completed for patient samples and the resulting regression revealed good agreement.A standardizes C-peptide measurement protocol was also established.This assay provides highly accurate and comparable results across testing systems and laboratories.Conclusion:In our study,two highly easy-assay based on GO were successfully developed and applied for detection of pathogen DNA and determination of serum C-peptide,separately.The purpose of this study was to establish LAMP-GO-LAPS assay as a rapid and bedside mycoplasma genitalium detection technique and develop GO-MS as a rapid,high throughput C-peptide measurement assay as a clinical reference method.Consequently,the graphene oxide not only shows the great potential in the detection of biomarkers and biomoleculars,but also opens up broad application prospects for biological sensors and MALDI-TOF MS.