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國立陽明交通大學 分子醫學與生物工程研究所 李明家所指導 張齡元的 開發Pluronic® F127/ Nichigo G-Polymer™ (G-Polymer)生物墨水應用於3D列印 (2021),提出SOL SS-2關鍵因素是什麼,來自於3D列印、皮膚組織再生、新型乙烯醇聚合物(Butendiol Vinyl Alcohol co-Polymer、Nichigo G-polymer™)、聚氧乙烯聚氧丙烯醚三嵌段共聚物(Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymer、Pluronic® F-127)、螺旋二十四面體(Gyroid)。
而第二篇論文長庚大學 化工與材料工程學系 劉繼賢所指導 Pravanjan Malla的 電化學結合免疫磁性粒子與奈米複合物碳電極之副甲狀腺素和病毒棘蛋白偵測 (2021),提出因為有 副甲狀腺素、網版印刷電極、多壁奈米碳管、磁性粒子、新型冠狀病毒的重點而找出了 SOL SS-2的解答。
最後網站Swami Shraddhanand College則補充:This college was established in 1967 to copiously cater to the education needs of students of North Delhi and adjoining areas at affordable cost and convenience ...
開發Pluronic® F127/ Nichigo G-Polymer™ (G-Polymer)生物墨水應用於3D列印
為了解決SOL SS-2 的問題,作者張齡元 這樣論述:
摘要 iAbstract ii致謝 iv目錄 v表目錄 ix圖目錄 x第一章 1緒論 11.1皮膚簡介 11.2皮膚癒合階段 21.3淺層皮膚傷口治療方法 41.4深層皮膚傷口治療辦法 51.5微結構與細胞生長 81.6 3D列印 91.7 Nichigo G-Polymer™ (G-Polymer) 101.8 Pluronic® F-127 121.9表皮生長因子 121.10 2D與3D培養 13第二章 14研究動機與實驗目的 14第三章 17研究方法與實驗步驟
173.1材料製備及分析 183.1.1 Gel permeation chromatography(GPC)量測 183.1.2 Pluronic® F127/ Nichigo G-Polymer™材料的改質與製備 18 3.1.3黏度測試(Viscosity) 193.1.4 IR測量與分析 193.1.5 Pluronic® F‑127/ Nichigo G-Polymer™ (G-Polymer)的3D列印 193.2體外細胞培養 193.2.1細胞的培養與接種
203.2.2列印螺旋二十四面體(Gyroid)的體外培養 203.2.3細胞毒性檢測 203.3統計分析 213.4細胞行為分析 213.4.1冷凍包埋(Optimal Cutting Temperature Compound, OCT)及切片 213.4.2蘇木素-伊紅染色(Hematoxylin and eosin stain, H&E stain) 21第四章 22 實驗結果與討論 224.1材料製備與檢測 224.1.1 Nichi
go G-Polymer™ (G-Polymer)的GPC結果 224.2 2D體外細胞實驗 214.2.1 Polyvinyl alcohol (PVA)和Nichigo G-Polymer™ (G-Polymer)的生物相容性比較 224.2.2 Nichigo G-Polymer™ (G-Polymer)生物相容性檢測 234.3材料改質&探討 244.3.1材料的改質 244.3.2改質後材料Nichigo G-Polymer™ (G-Polymer)/Pluronic® F127黏度測試 274.3.3 Pluronic® F‑127/ Ni
chigo G-Polymer™ (G-Polymer)生物毒性檢測 284.3.4 20w% F127 + 5w% G-polymer物理交聯後IR光譜變化 294.3.5 20w% F127 + 5w% G-polymer化學交聯後IR光譜變化 314.4 3D體外細胞實驗 324.5蘇木素-伊紅染色(Hematoxylin and eosin stain, H&E stain)結果 33第五章 35結論 35第六章 37 未來工作 37第
七章 38參考文獻 38
電化學結合免疫磁性粒子與奈米複合物碳電極之副甲狀腺素和病毒棘蛋白偵測
為了解決SOL SS-2 的問題,作者Pravanjan Malla 這樣論述:
CONTENTSChinese Abstract.………...…………………………………...………...……...……iEnglish Abstract ……………………………………………...……………………iiiContents……….……………………………………………...…………....…...…..vList of figures….……………………………………………...……………….…..xiList of tables…...……………………………………………......…………....…. xxiList of abbreviations……………………...………………
…...……..…...……..xxiii1. CHAPTER 1- INTRODUCTION………….……..…….…..11.1 Parathyroid hormone……….…..…….…….…..11.1.1 Synthesis and degradation……….…..……….……….….….21.1.2 Secretion and regulation……….…………...…..…31.2 PTH assay…………………..….…...……..51.2.1 First-generation PTH assay……………..….…61.2.2 Second generation
assay (intact PTH assays) ……….……..…71.2.3 Third generation assay (Bioactive PTH (1-84) assay) ………,….…..…...71.3 Clinical uses…………..……………...……81.4. Electrochemical sensor……………………91.4.1 Biosensor……….………………..….......91.4.2 Immunosensor……….……………….……….91.4.3 Enzyme immunoassay or enzyme-linked immunosorb
ent assay (ELISA)….101.4.4 Point-of-care test (POCT) ……….………….…….……101.5 Screen-printed carbon electrode…………….…111.6 Electrochemical measurement technique ………….….…121.6.1 Cyclic voltammetry……….…………….……..…….…131.6.2 Differential pulse voltammetry……….…….…….…....……131.6.3 Square wave voltammetry……….………
….…….…...……141.6.4 Electrochemical impedance spectroscopy……….……..……151.7 Nanomaterials……….………………..…….…181.7.1 Carbon-based nanomaterials……….…………....……..……201.7.2 Magnetic nanoparticles……….……………..…....211.8 Novel Coronavirus……………….….….…..…221.9 Goal of the work……………………....…232. CHAPTER 2- MATERIALS A
ND METHODS………….24PART-1 (Label-free parathyroid hormone immunosensor using nanocompositemodified carbon electrode).……..……...……...……….242.1 Chemical and reagents……….……………..…242.2 Electrodeposition of MWCNT-AuNP on SPCE working surface….……262.3 Immobilization of the immunosensor……….……………262.4 Calibrat
ion of PTH……….………………..……..…272.5 Characterization of SPCE electrochemical properties……….…….….…272.5.1 Measurement of electron transfer rate constant (Ks) by CV and EIS….…...272.5.2 Calculation of effective surface area……….………….....…282.5.3 Impedance analysis by EIS……….……………...….…292.5.4 EIS estimati
on of association constant between PTH and antibody……...…302.6 Statistical analysis……….…………….……...….…30PART-2 (Electrochemical immunoassay for serum parathyroid hormone usingscreen-printed carbon electrode and magnetic beads) ………...…..312.7 Modification of MBs………………….….……312.8 Fabrication of the ele
ctrochemical immunosensor……….……...…….…312.9 Optimization of HRP dilution and antibody concentration……….…322.10 Optimization of analytical procedure and signal recording…….........….33PART-3 (Voltammetric biosensor for coronavirus spike protein using magnetic beadand screen-printed electrode for poin
t-of-care diagnostics) ……….342.11 Immobilization of MB-APBA-Ab-HRP……….…….....……342.12 Detection of the COVID-19 spike protein……….………...…352.13 Optimization procedure and signal definition……….……….……352.14 SWV and EIS measurements procedure……….……….……362.15 Colorimetric assay……….…………….......…373. CHAPT
ER 3-RESULTS AND DISCUSSION……..……….…38PART-13.1 Characterization of prepared SPCEs……….………...…….…….…383.1.1 Microscopic surface characterization by FE-SEM……….…….…383.1.2 Transmission electron microscope and Raman spectroscopy…….……413.2 Electrochemical characterization of SPCE modification……….…….…443.
3 EIS characterization of modified SPCEs………….......…473.4 Effects of capture antibody concentration on PTH detection……...….…493.5 PTH detection using nanocomposite modified immunosensor…….……513.5.1 Estimation of association constant between the antibody and PTH antigen onSPCE……….……………………...……513.5.2
PTH detection using the EIS method……….…………..……513.5.3. PTH detection using the CV method………….….……533.6 Interference assay and storage stability of the immunosensor…….…..…55*Summary of Part-1…………………….……59PART-23.7 Characterization of modified MBs……………..……...…593.8 Parameter optimization of SWV measu
rement……….…….…623.9 Optimization of antibody and HRP concentration using SWV……….…643.10 Electrochemical characterization of modified MBs…………693.11 PTH detection using SWV and EIS methods………….….…703.12 Interference test……………………733.13 Storage and stability test………………...……743.14 Electron transfer rate
constant of MB-APBA-HRP-Ab……..….…77*Summary of Part-2………………......…….78PART-33.15 Principle of SARS-CoV-2 biosensor……….…....…..…793.16 Characterization of modified MBs……………….…..…803.16.1 Microscopic characterization of modified MBs……………803.16.2 Thermogravimetric analysis and magnetic hysteresis of MB
-NH2 andMB/APBA……………………….……..……833.16.3 Electrochemical characterization……………..…843.17 Detection of Spike protein……………...…..…..…873.18 Interference test…………………..…......923.19 Stability and storage test…………………..….923.20 Optimization of the experimental parameter……….…..…....943.20.1 Optimization of HR
P and antibody dilution…………….....943.20.2 Optimization of incubation time and H2O2/HQ concentration.....................953.20.3 Optimization of the MB volume and reaction time………….…963.20.4 Optimization of blocking reagent………………...….964. CHAPTER 4-CONCLUSION……………….…..…100REFERENCE …………………...…….101A
PPENDIX……………...……………..…..……114A1. Experimental section…….…………..…114A1.1 DNA sequences………………….……114A.1.2 Apparatus………………………..…...114A1.3 Synthesis of graphene oxide………………..115A1.4 Reduction of graphene oxide…………….......…..116A1.5 Synthesis of Au-r-GO-MNP-COOH……………...…..116A1.6 Modification of Au-r-GO
-MNP…………………116A1.7 Detection of COVID-19……………………117A1.8 Process of electrochemical measurement……………..117A1.9 Optimization of measuring parameters and signal definition……118A2. RESULTS………….……………...……...…119A2.1 Characterization of material…...………………120A2.1.1 SEM characterization………………...………..120A2.1.
2 Electrochemical characterizations………...……......121A2.2 Optimization of experimental parameter…………..…123A2.3 Specificity test of target DNA sequence……….….….124A2.4 Analytical performance of DNA sensor ………….….124Summary…………………………...….127Curriculum vitae ……………………...……128List of FiguresFig 1.1 Structu
re and composition of human parathyroid hormone …….….……...…8Fig 1.2 Overview of screen-printed electrode………...….….….…12Fig 1.4 (A) Potential waveform and (B) typical differential pulse voltammetry.…14Fig 1.5 Typical waveform of square wave voltammetry………….16Fig 1.6 Schematic diagram showing componen
t of AC of impedance……….16Fig 1.7 Randles circuit model for EIS analysis……….………17Fig 1.8 Electrochemical impedance spectra for electrochemical immunoassay forPTH detection……….……...………………..19Fig 1.9 Schematic illustration of the classification of nanostructure materials……20Fig 1.10 An overview of nan
omaterials application in different fields. ……..……23Fig 1.11 Structure of novel coronavirus SAR-CoV-2……..…….…...…23Fig 2.1 Schematic representation of fabrication of PTH immunosensor….…28Fig 2.2 Schematic diagram showing the fabrication process and the immunoelectrochemical reaction of magneto immun
osensor. ……….……….…33Fig 2.3 Schematic illustration for SARS-CoV-2 Spike protein detection using MBbased electrochemical biosensor. ……….……………...……35Fig 2.4 Schematic representation of S/B calculation.……….……….…37Fig 3.1 FE-SEM image of bare and modified SPCEs: (A) Bare, (B) AuNP/SPCE, (C)MWCNT-AuNP/SP
CE, with 1.0µm scale bar. Energy dispersive X-Rayspectroscopy results from the samples: (D) Bare SPCE, (E) AuNP/SPCE, (F)MWCNT-AuNP/SPCE.……….……………..……39Fig 3.2 FTIR spectra of bare SPCE (A), MWCNT/SPCE (B), AuNP/SPCE (C) andBSA/SPCE(D).……….…………..……….…40Fig 3.3 The microscopic images of the SPCE surf
aces by metallurgical microscope:(A) bare SPCE, (B) AuNP/SPCE, (C) MWCNT-AuNP/SPCE. Contact anglemeasurement on the modified SPCE surfaces: (D) bare SPCE, (E) AuNP/SPCE, (F)MWCNT-AuNP/SPCE……….……………...……41Fig 3.4 TEM images of nanocomposites in-situ synthesized on SPCE. (A) MWCNT,(B) MWCNT- AuNP, (C)
the size distribution of the prepared AuNP on SPCE usingthe deposition potential of -200 mV for 300 sec. (D) Raman spectra of bare SPCE,AuNP/SPCE, MWCNT-AuNP/SPCE. The scale bar was 100nm. ……….……………………….……43Fig 3.5 Chronoamperometric diagrams for assessing effects of deposition potentialusing 10 mM
of HAuCl4 (A) and effects of HAuCl4 concentration at -200mV (B) forelectrochemical deposition on SPCEs for 300 sec. In the inset, the linear response isshown by fixing the potential -200 mV for 300 sec.……….………44Fig 3.6 (A)Cyclic voltammogram and (B) ΔEp of SPCEs deposited bynanocomposite using diff
erent MWCNT-AuNP concentrations. Differentconcentrations of MWCNT (10~100 μg/mL) in 10 mM of HAuCl4 solution weretested at -200 mV for 300 sec. ……….……………….…45Fig 3.7 (A) Cyclic voltammogram of MWCNT-AuNP/SPCE measured in potentialwindow of -500 to +600 mV vs. Ag pseudo-reference electrode at differe
nt scanrates (from inner to outer) :10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mV s-1. (B) effectof scan rate on the anodic and cathodic peak current. (C)linear sweep voltammogram(LSV) of bare SPCE, AuNP/SPCE, MWCNT-AuNP/SPCE. (D) peak current andIpa/Ipc ratio of different SPCEs by LSV using 3mM ferric
yanide inPBS. …………………………46Fig 3.8 (A) Impedance results of different modified SPCEs using 3mM ferricyanidein PBS. EIS was run with amplitude 100 mV with amplitude 100mV, frequencyrange 1-1000 Hz, initial potential 50 mV. (B) Histogram of charge transfer resistanceof different SPCEs including bare SP
CE, MWCNT-AuNP/SPCE, Ab1-MWCNTAuNP/SPCE, FBS-Ab1-MWCNT-AuNP/SPCE, PTH-FBS-Ab1-MWCNTAuNP/SPCE. Human serum spiked with 100 pg/mL of PTH was used. Error barrepresents the standard deviation from three repeats (n=3). ……..….…50Fig 3.9 (A) EIS effect of capture antibody loading on the biosensor and Rct s
ignal(B). Effect of coating duration on EIS (C) and Rct signal (D) using 110 ng/mLantibody. Different antibody dilutions (1110, 222, 110, 55 ng/mL) were used toimmobilize the SPCE surface. The impedance spectra were obtained using 100pg/mL PTH concentration in all these experiments. Error bar repres
ents the standarddeviation from three repeats (n=3). …………….…51Fig 3.10 (A) Impedance spectra, (B) standard curve of PTH dosage (1~300 pg mL1) in human serum using the immunosensor. Impedance results for the measurementof the association constant using human serum spiked with PTH (C). (D) Cyclicvolta
mmetry current signal, (E) standard curve of PTH dosage (0~300 pg/mL) inhuman serum. The EIS and CV signal was obtained by placing 100 µL of3mM potassium ferricyanide in PBS. EIS was run with amplitude 100 mV,frequency range 1-1000 Hz, and initial potential 50 mV and CV run with potentialrange (-0.5
~0.6V) with 50 mV s-1scan rate. Error bar represents the standarddeviation from three repeats (n=3). ……….…………….55Fig 3.11 (A) Impedance spectra of immunosensor under interfering compounds at 1mg/mL, (B) histogram showing the relative Rct value. EIS was run with amplitude100 mV, frequency range 1-100
0 Hz, and initial potential 50 mV. The PTHconcentration in the interference test was 100 pg/mL. Error bar represents thestandard deviation from three repeats (n=3) …………….……..…58Fig 3.12 Stability test using fabricated immunosensor after 36-day storage: (A) CVsignal, (B) relative maximal current. The
current of Day-0 SPCE was represented as100%. (C) EIS signal, (D) relative Rct. The Rct of Day-0 SPCE was represented as100%. CV was performed over −500~+600 mV at 50 to 100 mV s -1using 3mMferricyanide in PBS. EIS signal was obtained by placing 100 µL of 3mM ferricyanidein PBS. EIS was run with am
plitude 100 mV, frequency range 1-1000 Hz, and initialpotential 50 mV. The tested PTH concentration was 100 pg/mL. * indicatedsignificantly different compared to Day-1 result at 5% level using a one-tailed t-test.The peak current (14.15 ±1.96 μA) of Day-1 SPCE was represented as 100% for theCV signa
l. The Rct (13496 ± 251 Ω) of Day-1 SPCE was represented as 100% forthe EIS signal. Error bar represents the standard deviation from three repeats(n=3). …………………………...…59Fig 3.13 SEM images of (A) pure APBA, (B) MB, (C) MB-APBA conjugate, with2.0µm scale bar…………………………61Fig 3.14 (A) Amount of APBA ads
orbed during conjugation of MB and APBA, (B)TMB assay of different dilution of HRP with MB-APBA. ………63Fig 3.15 (A) FTIR characterization, (B) TGA analysis of MB, and MBAPBA…………………………...…63Fig 3.16 Parameter optimization of SWV (A) increment time (B) pulse period. Theincrement range (5-20 ms) and pul
se range (100-500 ms) were tested. The SWVsignals were obtained using 0 and 100 pg mL-1 PTH concentrations. ΔI100 wasconsidered as signal and ΔI0 was blank. S/B ratio = ΔI100/ΔI0,n=3. …………………………….…64Fig 3.17 Optimization of (A) HRP dilution, (B) antibody loading on MB-APBA.SWV was run with amplitude
75 mV, pulse period 100 mV, and potential range (200mV~-400 mV). The antibody concentration 250ng mL-1 was selected during theHRP test (6A) and 200× HRP was selected for Ab-HRP optimization (6B). TheSWV signals were obtained using 0 and 100 pg mL-1 PTH concentrations. ΔI100 wasconsidered as signal
and ΔI0 was blank. S/B ratio = ΔI100/ΔI0,n=3. …………………………….…65Fig 3.18 (A) Optimization of incubation time of antibody and (B) hydroquinone andhydrogen peroxide concentration. n=3. ………...………65Fig 3.19 Optimization of kinetic parameter (A) effect of MB volume and (B)Michaelis-Menton constant Km. Effec
t of MB volume on Kcat and Km wasevaluated using 250 ng mL-1antibody loading. Different MB volumes (1, 3, 5, 7µL)were used to load on the working surface of SPE. ……………68Fig 3.20 Evaluation of different blocking reagents using (A)SWV signal and (B)DPV signal. All the blocking reagents are at a concen
tration of5%.…………………………...……69Fig 3.21 (A) Optimization of incubation time of MB. SWV was run with amplitude75 mV, pulse period 100 mV, and initial potential (200 mV~-400 mV). The SWVsignal was obtained using 100 pg mL-1 PTH concentration in this experiment. Errorbar represents the standard deviatio
n from three repeats (n=3). ……...…69Fig 3.22 (A) SWV currents of different MBs in the potential range (200 mV~-400mV), (B) histogram of SWV peak current using different MBs, (C) Impedancespectra of different MBs on bare SPEs (D) histogram of charge transfer resistanceusing different MBs. SWV was run
with amplitude 75 mV, pulse period 100 mV,and potential range (200 mV~-400 mV) using 5mM HQ+H2O2. Nyquist plots wererecorded on bare SPEs by 3 mM ferricyanide in PBS with amplitude 100 mV,frequency range 1~1000 Hz, initial potential 50 mV. Human serum spiked with 100pg mL-1 of PTH was used. n =3. …
………….…………71Fig 3.23 (A) SWV responses of the proposed immunosensor in differentconcentrations of PTH in 0.1 M pH 7.0 PBS containing 5mM HQ+H2O2, scanningfrom 200 mV to -400 mV with an amplitude of 75 mV s−1, a pulse period of 100msand (B) Calibration curve for PTH determination. (C) Nyquist curves r
ecorded in asolution of 0.1 M PBS containing 3 mM ferricyanide with applied potential was 0.05V at the frequency range of 1-1000 Hz and (D) EIS standard curve ofPTH…...………………………...…73Fig 3.24 SWV histogram of the fabricated immunosensor (MB-APBA-HRP-AbPEG) under the interfering compounds at 1mg mL -
1. The tested PTH concentrationin human serum was 100 pg mL-1. n =3. …………….………75Fig 3.25 Stability test of the immunosensor after 35-day storage at 4°C. SWV wasrun with amplitude 75 mV, pulse period 100 mV, and initial potential (200 mV~-400 mV) using 5mM HQ+H2O2. The tested PTH concentration was 10
0 pg mL1. …………………………….……77Fig 3.26 (A) Cyclic voltammograms (CV) of MB-APBA-HRP-Ab on SPE, (B) CVof SPE alone, (C) linear relationship of the square root of scan rate on the anodicpeak currents using 3 mM ferricyanide in PBS. CV measured in potential windowof -500 to +600 mV vs. Ag pseudo-reference
electrode at different scan rates (10,30, 50, 70, 100 mV s-1). ……………………..……78Fig 3.27 Schematic illustration for Spike protein detection using the MB-basedelectrochemical immunosensor.……………….……..…81Fig 3.28 SEM images of (A)MB-NH2, (B)MB/APBA, (C)MB/APBA/Ab-HRP,(D)MB/APBA/Ab-HRP/GLU/Spike protein wi
th scale bar: 1 µm, (E) sizedistribution of modified MBs…………………...…82Fig 3.29 Comparison of (A)APBA conjugated on two kinds of MBs, (B) horseradishperoxidase activity on two kinds of MBs using TMB assay. ……….….……83Fig 3.30 TEM images of (A)MB-NH2 (B)MB/APBA (C)MB/APBA/Ab-HRP(D)MB/APBA/Ab-HRP/GLU/Spi
ke protein with 0.5µm scale bar……..……..….84Fig 3.31 (A) Thermogravimetric analysis of modified MBs, (B) magnetic hysteresisof MB-NH2 and MB/APBA…………………...……..…85Fig 3.32 (A) Cyclic voltammogram of different MBs during modification. (B) peakcurrents and Ipa/Ipc ratio of different MBs on SPEs by using
3 mM ferricyanide inPBS. CV measured in the potential window of -500 to 600 mV using Ag pseudoreference electrode at a scan rate of 50 mV s-1. (C) impedance results of differentmodified MBs using 3 mM ferricyanide in PBS. EIS was run with amplitude 100mV, frequency range 1~1000 Hz, initial potentia
l 200 mV. (D) histogram of chargetransfer resistance of different MBs including PBS, MB-NH2, MB/APBA,MB/APBA/Ab-HRP on bare SPE. Error bar represents the standard deviation fromthree repeats (n =3). …………........................................................87Fig 3.33 Square wave voltammetry signal
s generated from SARS-CoV-2 Spikeprotein spiked in (A) saliva, (B) urine, (C) serum by varying Spike proteinconcentrations. Calibration curves based on peak currents from (D) saliva, (E) urine,(F) serum using 30-min incubation of Spike protein with MBs. Each data pointrepresents the mean ± SD of thr
ee separate measurements obtained using the sameSPE. Detection was carried out on the working surface of SPE by placing an externalmagnet by loading 5 µL of sample and 5 seconds of accumulationtime. ……………………….....…...89Fig 3.34 Standard curve of Spike protein concentration (3.125~200 ng mL−1) insali
va using the ELISA kit. …………………...…...91Fig 3. 35 Comparison of standard curves of MB-based electrochemical biosensorand the colorimetric TMB assay. The spike protein dissolved in humanserum. ………………………..…....91Fig 3.36 (A)Impedance results for the measurement of the association constant usingSpike p
rotein (B) standard curve of △RCT(Ci)/RCT(C0) and Spike proteinconcentration. ………………………..……....92Fig 3.37 (A)Impedance results for the measurement of the association constant usingSpike protein (B) standard curve of △RCT(Ci)/RCT(C0) and Spike proteinconcentration. ……………………..…....93Fig 3.38 Stability
tests of (A) MB/APBA and (B) MB/APBA/Ab-HRP/GLU after49-day storage at 4°C. SWV was run with amplitude 75 mV, pulse period 100 mV,and initial potential (-400 mV ~ 200 mV) using 5 mM H2O2/HQ. The tested Spikeprotein concentration was 10 ng mL -1. ……………..….…...94Fig 3.39 Optimization of (A) HRP fold,
(B) antibody dilution, (C) incubation timeof antibody and MBs, (D) ratio of hydroquinone and hydrogen peroxide, (E) MBloading volume, (F) reaction time of MB with H2O2/HQ. SWV currents weregenerated from serum samples spiked with Spike protein at 0 and 10 ng mL-1 andcorresponding S/B ratios using i
mmunosensors. ΔI10 was considered as signal andΔI0 was blank. S/B ratio = ΔI10/ΔI0, n=3………………..……97Fig 3.40 Evaluation of blocking agents on the signal of signal-to-blank ratio usingthe SWV methods (n =3). The antibody concentration 100× and 100× HRP wereselected for this test. The SWV signals were
obtained using 0 and 10 ng mL-1 Spikeprotein concentrations. ΔI10 was considered as a signal and ΔI0 was a blank. S/B ratio= ΔI10/ΔI0, n=3. All the blocking reagents are at a concentration of 5%..................90Fig 3.41 (A) Optimization of APBA concentration, the (B) effect of antibody-HRPwith TM
B on activity, (C) effect of Ab-HRP incubation time, (D) optimization ofincubation time of Spike protein…………………100Fig A1 Schematic illustration of the electrochemical genosensor for the detection ofSARS-CoV-2……………………….…119Fig A2 SEM image of (A) graphene. (B) r-GO, (C) r-GO-MNP-COOH (D) r-GOMNP-COOH
(E) MNP-COOH and (F, G, H, I, J) relative EDX spectrumrespectively. All the images were taken with a scale bar of 2.00 µm………120Fig A3 (A) Raman spectra analysis of MNP-COOH, r-GO-MNP, Au-r-GO-MNP.(B) thermal analysis of MNP-COOH, r-GO-MNP, Au-r-GO-MNP………...121Fig A4(A) Cyclic voltammetry step-by-st
ep modification of MNP (B) the bardiagram represents relative peak current, (C) electrochemical impedancespectroscopy during each step modification, (D) relative Rct value. All theelectrochemical measurement was performed by using Autolab. Where (a) representbare, b) Au-r-GO-MNP, c) Au-r-GO-MNP/N2R-
NH2/BSA, d) Au-r-GO-MNP/N2RNH2/BSA/N2R+N3R/N3-biotin/HRP respectively………….122Fig A5(A) Optimization of captured sequence N2R-NH2 concentration (B) detectionsequence N3R-biotin concentration (C) incubation time of hybridization….123Fig A6(A) Comparison of specificity of designed genosensor with ds-DN
A and ssDNA. (B) comparison of non-target GFP and ss-DNA………….124Fig A7(A) Square wave voltammetry response of genosensor to differentconcentration of target complementary sequence (5*10-9~5*10-2 nM) in humansaliva, (B) the linear calibration curve plotted of peak current vs. the logarithm ofthe ss-D
NA concentration (C) SWV response different concentration ofcomplementary sequence dissolved in human urine, (D) relative standardcurve………………………….….125List of tablesTable 1 Chemicals and reagents list……….…………...…….25Table 2 Apparatus list…………………..…….26Table 3 Electrochemical and physicochemical char
acteristics of the modifiedSPCEs ……….……………………47Table 4 Determination of Rs, Rct, Cdl from EIS signal shown in Fig 3.8 usingZsimpwin software………………….…….…49Table 5 Recovery of this impedimetric and amperometric immunosensor versusELISA in actual sample…………………...…….…53Table 6 Comparison of the proposed
PTH immunosensor performance with theprevious methods……….……………………56Table 7 Summary of zeta potential and size of modified magnetic beads……62Table 8 Summary of optimization of experimental parameters…………67Table 9 Comparison of recovery of PTH by ELISA, SWV and EISmethods……………………….…...74Table 10 Comp
arison different PTH immunosensors in literature……….….76Table 11 Zeta size analysis of MBs……………….……83Table 12 Electrochemical characteristics of the modified MBs on bare SPE.…....88Table 13 Limit of detection of MB-based electrochemical biosensor using Spikeprotein spiked in different body fluids ……
…………...90Table 14 Comparison of recovery yield of Spike protein by SWV, EIS, and ELISAmethod………………………..…...92Table 15 Comparison of various nanomaterial-based electrochemical methods fordetection of SARS-CoV-2…………………..…….95Table 16 Summary of optimization of experimental parameters……..…99Table A1 Li
st of DNA primer sequences used in this work…….…….114Table A2 Comparison of size modified MNP captured with DNA sequence…….124Table A3 Comparison of Ks of different modified MNP captured with DNAsequence……………………….…124Table A4 Limit of detection of Geno sensor using in different bodyfluids……………………………
.124
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