The emission spectra of the preparing FRET fluorescence pairs has been presented in Figure 4-3.75 The PDDA-QDs sizes were chosen to maximize the spectral overlap of the donor-acceptor optical spectra. The detail study of PDDA-QDs has been reported in reference. 75
Figure 4-3. Normailized spectra of (a) emission of PDDA-QDs (530nm) (b) emission of PDDA-QDs (543nm) (c) absorption of CA/TTD/TMR (d) emission of CA/TTD/TMR (570 nm) at excited wavelength 400 nm. All spectra were measured in PBS system (20mM, pH 7.5).
The fundamental research of bio-component KSI protein has been described in chapter 2. KSI protein can easily bind with hydrophobic chemical component such as cholic acid due to intra-molecular hydrophobic interaction. The following PL
spectrum (Figure 4-4 and Figure 4-5) of different ratio of PDDA-QDs (donors) and CA/TTD/TMR (acceptors) were utilized to explore the efficiency of FRET with or without KSI.
Assays examining the FRET efficiency of the various pairs were carried out by serial titration of acceptor fluorophore, which were made to produce a range of average QDs only: CA/TTD/TMR and QDs/KSI:CA/TTD/TMR ratios ranging from 1:0.3 to 1:24. In this study, KSI exhibited an ability to keep the suitable distance between donor and acceptor by protein-small molecule binding and the stronger FRET efficiency has been shown in Figure 4-4.
(A)
(B)
(C)
Figure 4-4. (A) The concentration of QDs/Y14only_KSI was kept fixed and the concentration of CA/TTD/TMR was increased from 0 to 8 µM at pH 7.5, 20 mM phosphate buffer. The decreasing fluorescence intensities at 530 nm, which was presented the intramolecular interaction between Y14only_KSI and CA/TTD/TMR by
FRET. (B) The normalized spectra has presented the efficiency of FRET by QDs/PDDA quench. The KD value of CA/TTD/TMR was measured (at
λ
530 nm)
to be 0.6 µM by the quench yield of QDs/PDDA. (C) The FRET efficiency has been normalized to measure by the increasing fluorescence intensities of CA/TTD/TMR (atλ
570 nm).
In Figure 4-4B, which shows substantially violent fluorescence intensities change of the CA/TTD/TMR emission (at
λ
570 nm)
due to FRET. The sensitive emission specta has been normalized from Figure 4-4A and the control experiment has been shown in Figure 4-5B. On the other hands, we observed a progressive and substantial QDs populations quench in the energy transfer efficiency with increasing number of CA/TTD/TMR attached to a single nanocrystal. The resulting normalized QDs emission intensities were plotted versus the acceptor concentrations in Figure 4-4C and Figure 4-5 C. The comparing results indicated the nonspecific interaction between donors and acceptor also can influence the strength of QDs quench. However, in Figure 4-4C, the analyte-binding protein immobilized on the core-shell surface, which was caused the stronger contact-quenching of the QDs by FRET. And the binding constant of CA/TTD/TMR has been measured to be KD= 0.6 µM.(A)
(B)
(C)
Figure 4-5. (A) PL spectrum of the QDs/PDDA (530 nm) donors and CA/TTD/TMR (570 nm) dye acceptors for each ratio in titration series CA/TTD/TMR without biocomponent KSI involving. (B) The normalized spectra presents the efficiency of FRET by QDs/PDDA quench. (C) The FRET efficiency has been normalized to measure by the increasing fluorescence intensities of CA/TTD/TMR (at
λ
570 nm).
4-4. Conclusion
Cholic acid FRET-based biosensing platform have been demonstrated by the utilization of QDs/PDDA (donors) and CA/TTD/TMR (acceptors), due to the emission spectra of QDs achieves high energy transfer efficiency with organic dye.
The stronger FRET efficiency has been presented as KSI protein involves in this system. On the other hands, the measurement of the binding constant of CA/TTD/TMR (KD= 0.6 µM) was also derived from FRET efficiency of QDs/PDDA.
were dissolved in 10 ml dry DMF on ice bath for 30 mins. DIPEA (1 ml, 6mmole) was dropped slowly into the reaction mixture. The reaction mixture was stirred at RT overnight.
Chapter 5
Conclusion
This novel steroid-sensing model was constructed by intra-molecular binding of a fluorophoric ligand, 5-(2-aminoethylamino)-1-naphthalenesulfonate (1,5-EDANS) moiety, through an alkyl linker bonded to a residue in the proximity of the steroid-binding site of the sensing protein. This fluorophoric ligand was successfully synthesized and identified by MALDI-TOF analyzer. The protein used for steroid recognition was derived from a genetically engineered △5-3-ketosteroid isomerase (kcat/Km = 1.12x107 (M-1s-1) and further conjugated uniquely with the ligand at its Cys-86. The major driving force favoring this association is generally thought to be the hydrophobic effect which prompted the hydrophobic ligand to bind with the protein. The hydrophobic effect has been demonstrated relied on the identification of the site of I-14 labeling which was confirmed by steady-state fluorescence and MS measurements.
The comparison of steady-state fluorescence spectra of various fluorophore-labeled proteins revealed that the emission characteristics varied with the environment where the ligand situated. The evaluation of fluorescence anisotropy decay of the fluorophore suggested the existence of the intramolecular protein-ligand binding interaction. For KSI/mA51 system, 25% of the linked mA51 was estimated to be inside the steroid-binding site, while 75% remained outside the binding cavity.
Our designed system further immobilized on the surface of a silicon nanowire. In the presence of a steroid, the negatively charged 1,5-EDANS moiety, which presumably occupies the steroid-binding site, is expelled and exposes to the nanowire surface. The electrical response produced from the 1,5-EDANS moiety is measured and the concentration is calculated accordingly. The sensitivity of this novel nano-bio-device
can attain a femtomolar level.
In order to improve the complex designed model for non-charge analytes detection on SiNW-FETs, a novel immunobioassay system has been developed. Open - sandwich bioassay has been designed, which employs the antigen dependency of the interaction between the separated heavy chain (VH) and light chain (VL) of an antibody variable region (Figure S5). Bisphenol A (BPA) detection was first to use for the application of SiNW-FETs by open - sandwich bioassay strategy. In our case, VL
firstly conjugated with BS3-activated silicon oxide surface and then introduced the analyte (0.334mg/mL BPA mixed with 1.126 µg/mL VH) into the system. The obvious conductance change was caused by strong interaction of open - sandwich bioassay, as we compare with the following control experiments. However, without antigen (BPA), it’s hard to get any conductance respond because the two fragments difficultly associated to perturb the charged density on the surface of SiNW-FETs. On the other hand, without antibody, the intrinsic limitation of FET restricts noncharged BPA detection on SiNW-FETs, which was not also induced any conductance respond.
Finally, Cholic acid FRET-based biosensing platform have been demonstrated by the utilization of QDs/PDDA (donors) and CA/TTD/TMR (acceptors), due to the emission spectra of QDs achieves high energy transfer efficiency with organic dye.
The stronger FRET efficiency has been presented as KSI protein involves in this system. The measurement of the binding constant of CA/TTD/TMR (KD= 0.6 µM) was also derived from FRET efficiency of QDs/PDDA.
Chapter 6
Supplementary
Figure S1. The chemical structure of mA51-mA51.
Figure S2. The measured molecular weight of mA51-mA51 is 734 ± 1 amu, which is consistent with the calculated value of 733 amu. The values of m/z = 146.2, 153.3, 314.4 were measured from matrix.
10000
146.2
153.3
314.4
734.1 756.2 0
8000
6000
4000
2000
100 200 400 500 600 700
Figure S3A. The level complementary of a pair of primers were designed to construct the gene of Art_KSI protein by PCR methods.
Figure S3B. (a) Agarose electrophoresis gel results of cloning. M, molecular markers;
lane 1, was first DNA fragment (~100 bp) amplified by PCR; lane 2, second DNA fragment (~200bp) amplified by PCR; lane 3, was third DNA fragment (~280 bp) amplified by PCR ; and land 4, was full length of DNA fragment (375 bp) amplified by PCR.
Figure S4A. The SiO2 surface morphology is very smooth and its roughness is the smallest in the three samples after the APTMS modification..
Figure S4B. The silanized SiO2 surface was caused increasing its roughness by using BS3 conjugation.
Figure S4C. The AFM images of the SiO2 surface with biomolecular treatment, which has the visible difference with the morphology of SiO2 surface that modified by BS3.
Chapter 7
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