Investigating a Carboxylated Terthiophene Surface for SPR P4 Detection
One of the crucial steps in development of biosensors is immobilization of bioreceptors on sensor surfaces. In terms of SPR detection, where gold surfaces are most commonly used, the surface density and orientation of bioreceptors as well as the distance or proximity of ligand binding sites to SPR gold surfaces play important roles in the sensitivity and reproducibility of assays. The immobilization is usually accomplished by covalent binding of bioreceptors to the chemical groups of coating reagents on gold surfaces . Thus, gold coating reagents are important in the robustness of bioreceptor immobilization as well as the stability and reusability of sensor surfaces. The examples of gold coating reagents are SAMs , carboxylated dextran , polypeptieds , thin films of methacrylic acid copolymer , mixtures of poly-L-lysine and titanium oxide  and graphene . Among them, a biocompatible dextran hydrogel matrix is one of the best so far, evidenced by its great utility in SPR analyses and commercial success as the Biacore chips. The success of the Biacore chips is mainly attributed to the high binding capacity, wide versatility, good reproducibility and high surface stability of the dextran matrix. However, the procedure to deposit such a carboxylated dextran hydrogel matrix is quite complicated and requires the use of aggressive and toxic reagents such as epibromohydrin, bromoacetic acid and strong alkali . The dextran hydrogel matrix also faces some challenges of non-specific interactions  and steric effects . Furthermore, the thickness of the dextran hydrogel  (100-200 nm for a CM5 chip) significantly reduces the actual SPR working range on gold surfaces from 300 nm to 100-200 nm, and also decreases SPR effects or sensitivity of assays by holding binding sites away from gold surfaces. Therefore, opportunities exist to investigate other coating reagents which form thinner layers on gold surfaces to increase the SPR effect, and at the same time, retain similar surface biocompatibility and stability, to replace the widely used carboxylated dextran hydrogel matrix on gold surfaces. Terthiophene (T3) has been investigated over the last two decades mainly for the synthesis of polyterthiophene, a conducting polymer which has special optical and electrical characteristics with wide application in antistatic materials, conductors, photovoltaic cells, electrode materials and organic semiconductors . Also polyterthiophene was used to develop biosensor surfaces, including electrode coatings for electrochemical biosensors [132-136] and MIPs for SPR biosensors . However, to our knowledge, the use of T3 itself in biosensors is limited, hence it is of our interest to explore the suitability and capability of T3 SAMs in coating gold surfaces for SPR detection. Previously, our group designed a new carboxylated T3 molecule (T3C, Figure 2.1) as a potential coating reagent for gold surfaces. The T3C molecule has multiple sulfurs in the thiophene rings that can adhere to a gold surface, and a pendent chain of a carboxylated oligo ethylene glycol (OEG) to potentially immobilize a bioreceptor which possesses a primary amine group.
Materials and Methods
Reagents and instrumentation
Phosphate buffered saline (PBS) tablets, ovalbumin (OVA), NaH2PO4.2H2O, Na2HPO4, N,Ndimethylformamide (DMF), N,N-Dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), NaOH, glycine, H2SO4, H2O2, Tween-20 and P4 were purchased from SigmaAldrich. N-Hydroxysuccinimide (NHS) was supplied by ACROS organics. T3C was provided by Digital Sensing Limited (DSL), New Zealand. 4-Pregnen-3, 20-dione 3-O-Carboxymethyloxime (P4 3-CMO) was supplied by Steraloids, Newport, USA. Anti-P4 monoclonal antibody (mAb) raised in mouse was purchased from AbD Serotec (7720-1430), BioRad. Plain gold chips, CM5 chips, an amine coupling kit, HBS-EP buffer and PD-10 desalting columns were supplied by GE Healthcare Life Science. All the SPR assays were conducted on Biacore Q system from GE Healthcare Life Science. The protein concentrations were determined by Nanodrop 2000 and the steroid conjugation degree was measured by matrix-assisted laser desorption/ionization mass spectrometry with time-of-flights (MALDI-TOF) on UltrafleXtremeTM, Bruker, USA.
Synthesis of P4-OVA
P4 3-CMO (19.4 mg) was dissolved in 0.4 ml of DCC/DMF (206.3 mg/ml) solution with stirring, followed by addition of 0.4 ml of NHS/DMF (115.1 mg/ml) solution. The reaction mixture was stirred at room temperature for 3 hours until white precipitates were formed. Then the mixture was added to a solution of OVA (44.4 mg) in PBS (4 ml, 0.2 M, pH7.4) as illustrated in Scheme 2.1. After stirring at 4°C overnight, the crude P4-OVA conjugates were dialyzed against water over 48 hours, followed by final dialysis against PBS (1.2 L, pH7.4) over 24 hours. The dialyzed product was further purified by passing a PD10 column. After dialysis and filtration, 7 ml of the P4-OVA conjugates solution was centrifuged at 10,000 rpm for one minute to remove white precipitates and the clean supernatant was collected. The aliquots were stored at -20oC. The protein concentration of P4-OVA conjugates was determined by Nanodrop 2000 and the steroid conjugation degrees were measured with MALDI-TOF.
Fabrication of T3C SAM SPR surfaces
Biacore SPR plain gold chips were cleaned with reactive ion etching (RIE) by March CS-1701 (Nordson MARCH, California, USA) using an oxygen plasma (set at 50 W for 30 seconds and 50% O2) for two minutes before the chips were immediately immersed in T3C (Figure 2.1) solution (10 mM, ethanol) overnight and thoroughly rinsed with ethanol (100%) and water. Then the chips were dried under nitrogen and assembled into the chip cassettes according to the manufacturer’s instructions.
Immobilization of P4-OVA
T3C SAM SPR chips and carboxylated dextran chips (CM5 chip) were activated by 150 µl injection of a mixture (1:1 v/v) of EDC (390 mM) and NHS (100 mM) at 5 µl/min, followed by 150 µl injection of P4OVA conjugates solution (1:20 v/v, acetate buffer pH 4.0) at 5 µl/min respectively. Then the chips were deactivated by a 100 µl injection of ethanolamine solution (1 M, pH 8.5) at 5 µl/min.
Antibody binding and inhibition assays of P4
A series of mAb solutions were prepared by diluting the stock solution in the PBS buffer containing Tween20 (0.005% v/v) to the desired concentrations. The dilution series of mAb used for P4-OVA that immobilized the T3C SAM SPR surface was 0, 0.2, 0.4, 0.6, 0.8 and 1 µg/ml; and that for the dextran hydrogel surface (CM5 chip) was 0, 0.8, 1.6, 2.4, 3.2 and 4 µg/ml. 60 µl of each mAb solution was injected at 20 µl/min over the two SPR surfaces respectively (Scheme 2.2). Surface regeneration was performed by injection of a 10 µl NaOH solution (10 mM) and glycine (10 mM, pH 2.0) for each. The injection of each mAb solution was repeated in triplicates. Inhibition assays were conducted by mixing a fixed amount of mAbs (0.8 µg/ml in the assays on T3C SAM SPR surface and 4 µg/ml on CM5 chip respectively) with a series of standard P4 solutions (1:1 v/v). The P4 standards used in the assays on a T3C SAM SPR surface were 0, 0.01, 0.1, 1, 5, 10 and 100 ng/ml; and those on the CM5 chip were 0, 0.1, 1, 5, 10, 20 and 100 ng/ml. After incubation for 30 min, 60 µl of each mixture was injected at 20 µl/min followed by the regeneration of the SPR surfaces as above. The injection of each mixture was repeated in triplicates. The results were analyzed statistically using Sigma Plot version 12.5 and all inhibition assay curves were fitted to 4-parameter logistic regression. The LODs were calculated as the concentrations corresponding to the blank signals less two standard deviations of the blank signals.
Results and Discussion
Synthesis of P4-OVA
P4-OVA conjugates were prepared by covalently binding the primary amine group of OVA and the carboxyl group of P4 3-CMO via carbodiimide reaction by DCC/NHS. The synthesis procedure also produced a byproduct dicyclohexylurea (DCU) (Scheme 2.3), which was insoluble in both organic solvents and aqueous buffers, and resulted in a white turbid mixture. To remove this byproduct together with excess reagents used for the reaction, dialysis, filtration and centrifugation were performed sequentially, which took about three days. The large amount of DCU formation was a result of excessive reagents used to ensure the conjugation efficiency of P4 and OVA, as P4 3-CMO N-hydroxysuccinimidyl esters had low solubility in the aqueous OVA solution. However, the presence of insoluble DCU in the conjugation reaction may impair reaction rates by affecting interaction of P4 3-CMO Nhydroxysuccinimidyl esters with OVA and it was difficult to completely remove DCU from the final product even with the use of dialysis, filtration and centrifuge. The impurity of the final P4-OVA conjugates may further affect immobilization to the sensor surface or the sensitivity of the assays. As an alternative, EDC may replace DCC to avoid an insoluble byproduct, but EDC has lower solubility in DMF and thus may be less efficient in activating P4 3-CMO. Still, the time-consuming purification by dialysis and filtration is not avoidable when P4-OVA is synthesized in solution. Another approach of synthesizing P4-OVA conjugation in situ on sensor surfaces was investigated. The conjugation degree of P4 moiety in the conjugates was determined by MALDI-TOF as shown in Figure 2.2. The molecular weight (MW) of the unmodified OVA was approximately 44,500 Da according to mass spectroscopic analysis (Figure 2.2a). The MW of two P4-OVA conjugates synthesized separately were 45,739 Da and 45,594 Da (Figure 2.2b, c). As the MW of P4 3-CMO moiety is about 370 Da, on the average, P4-OVA 1 and P4-OVA 2 had approximately 3.3 and 2.9 P4 moieties per OVA respectively. The concentrations of P4-OVA conjugates were measured by NanoDrop using a customized method which measured the absorbance of peptide bonds at 205 nm. The concentration of P4-OVA 1 and 2 was 3.75 mg/ml and 3.82 mg/ml respectively. Thus, the two separate syntheses of P4-OVA resulted in similar yields and degrees of conjugation.
1.Introduction to Progesterone (P4) Tests in the Reproductive Management of Dairy Cows and Biosensors
1.1 The Reproductive Management of Dairy Cows
1.2 P4 in Cows
1.3 The Methods of Measuring P4
1.4 Biosensing assays
1.5 Research Aims and Thesis Outlines
2. Investigating a Carboxylated Terthiophene Surface for SPR P4 Detection
2.2 Materials and Methods
2.3 Results and Discussion
3. Functionalizing Sensor Surfaces for P4 Detection by Surface in situ Synthesizing P4-OVAs
3.2 Materials and Methods
3.3 Results and Discussions
4. Developing Antibody and MB Conjugates Based on SPR Assays for Detecting Milk P4
4.2 Materials and Methods
4.3 Results and Discussions
5. Investigating a P4 Aptamers for the Replacement of anti-P4 Antibodies
5.2 Materials and Methods
5.3 Results and Discussions
6. Conclusions and Future Perspectives
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