Synthesis and structure investigation of stabilized aromatic oligoamides and their interaction with g quadruplex structures 6

Chapter Gold Nanoparticles-Based Colorimetric Detection of Proteins 6.1 Introduction Gold nanoparticles (AuNPs)-based molecular recognition approaches are emerging as attractive calorimetric probes by providing sensitivity and selectivity that are comparable to more conventional chromogenic sensors, such as fluorescence techniques. In particular, oligonucleotide-functionalized gold nanoparticles (DNA-Au NPs) have been used to develop many assays for a wide variety of analytes, including proteins,1-3 oligonucleotides,4-7 metal ions8-11 and other small organic molecules.12-15 One remarkable example is the use of aptamer-functionalized gold nanoparticles for selective molecular detection.16-19 Aptamers are oligonucleotides isolated to bind to a variety of molecular targets with high specificity and binding affinity, ranging from small organics to proteins.20-22 Since aptamers are selected using a relatively rapid in vitro selection process and can be inexpensively synthesized, using aptamer for specific molecule binding studies has drawn much interest recently.23-25 Adequate transducing elements are required to generate a physically measurable signal from the recognition process. The optical detections of aptamer-molecule interactions were reported using fluorescence26 or evanescent wave-induced fluorescence.27 However, 177 these methods involve a tagging process or sophisticated experimental techniques. Therefore, aptamer-functionalized gold nanoparticles have shown to be a powerful approach for molecular recognition combining the sensitivity of gold nanoparticles and the binding specificity of aptamers. The past few year have witnessed advances in the integration of functional aptamers into gold nanoparticles to generate hybrid sensors for specific and sensitive molecular recognition. The typical mechanism for these systems is analyte-induced cross-linking of AuNPs modified with molecules-binding aptamers, causeing color changes in solution as a result of electronic dipole-dipole coupling and scattering between neighboring Au particles. Dispersed AuNPs appear red due to the greater interparticle distance than their average particle diameter, while the aggregates change into purple as the interparticle distance decrease below the average particle diameter. Systems base on analyte-induced aggregation of AuNPs have been applied for the colorimetric detection of small molecules, metal ions and proteins. Many groups have carried out extraordinary research in this area. Lu’s group has done continuous work in the detection of adenosine and cocaine based on the crosslinking mechanism of AuNPs by hybridizing aptamers with complementary sequences.17-19 Mirkin et al have demonstrated the detection of ions (Hg2+) and cysteine12 using aptamer-functionalized gold nanoparticles with very high sensitivity and selectivity. However, previous work focused mostly on colorimetric detection of small molecules, whereas so far only a few studies34-36 reported the detection of proteins by 178 the integration of AuNPs with aptamers. The commonly employed protein for proof-of-concept assay development by aptamer-based AuNPs is thrombin which is involved in the blood clotting process. Since thrombin is coagulation protein that plays many roles in the coagulation cascade, thrombin-binding aptamer (TBA) is usually considered as an important pharmaceutical target when searching for anti-coagulants and antithrombotics to interfere with the blood coagulation. Two aptamers have been identified to selectively bind to human α-thrombin.28,29 15-mer TBA (5’-GGTTGGTGTGGTTGG-3’), which binds to the fibrinogen-binding site of thrombin with Kd ≈ 100 nM, and (5’-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3’), heparin-binding site of thrombin with Kd ≈ 0.5 nM). 29-mer which binds TBA2 to the Willner et al first reported the use of aptamer-functionalized AuNPs as a catalytic label for the amplified detection of thrombin in solution and on surfaces, with the detection limits around 20 nM and nM, respectively.30 In this system, nanoparticles were cross-linked by thrombin since each thrombin molecule binds two TBA aptamers. Recently, several aptamer-linked sandwich assays (duplex approach) were also designed for thrombin detection. These methods require complicated experiment techniques and sophisticated instruments, such as inductively coupled plasma-mass spectrometry,31 or electrochemical method.32 Herein, a novel method of double aptamer approach based on AuNPs to detect proteins were proposed and the detection of thrombin using gold nanoparticles modified with two different aptamers was systematically investigated. In addition, a simple duplex approach was also designed as a competitive method. 179 6.2 Result and Discussion 6.2.1 Preparation of Aptamer-based Gold Nanoparticles Table 6.1. DNA and DNA-modified AuNPs. Name Sequence Remarks TBA 5’- GGTTGGTGT GGTTGG-3’ Thrombin-binding aptamer TBA2 5’-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3’ Thrombin-binding aptamer Apt29R 5’- AGTCACCCCAACCTGCCCTACCACGGACT-3’ Complementary sequence of TBA2 Apt15R 5’-CCAACCACA CCAACC-3’ Complementary sequence of TBA Au-T1 Au-SH-3’-AAAAAAAAAACTAGGTTGGTGTGGTTGGTGTATC-5’ Modified TBA1 immobilized on gold nanoparticle Au-T2 Au-SH-3’-AAAAAAAAAACTA GTACA CCAACC-5’ Complementary sequence of modified TBA1 immobilized on gold nanoparticle Au-T3 Au-T4 Au-SH-3’-AAAAAAAAAACTATCAGTGGGGTTGGACGGGATGGTGCCTG Modified TBA2 immobilized on gold ATGTATC-5’ nanoparticle Au-SH-3’-AAAAAAAAAACTA GATACA TCAGGC-5’ Complementary sequence of modified TBA1 immobilized on gold nanoparticle Au-T5 Au-SH-3’-AAAAAAAAAACTA GGTTGGTGT GGTTGG-5’ TBA1 immobilized on gold nanoparticle Au-T6 Au-SH-3’-AAAAAAAAAACTA TBA2 immobilized on gold nanoparticle TCAGTGGGGTTGGACGGGATGGTGCCTGA-5’ All UV-Vis spectra are normalized against the spectrum of deionized water. A typical UV-Vis spectrum of aptamer-AuNP complex is shown in Figure 6.1. The Surface Plasmon Resonance absorption of the gold nanoparticles is evident from the red curve having its maximum absorption at 520 nm. This absorption spectrum is responsible for the reddish color of the gold colloid. In addition, the weak peak at 260 nm corresponds to the absorption from oligonucleotide. From the red curve and the difference curve after subtraction of red curve from the black one, we can deduce the concentrations of the gold particles and the aptamers from the absorbance using the Beer-Lambert law: Absorbance = Ecl 180 Where E is molar extinction coefficient, c is molar concentration, and l is path length. Given Eapt29 =143300 M-1cm-1 and EAu =105614000 M-1cm-1. From Figure 6.1, the concentrations of AuNPs and aptamer are 4.5 nM and 490 nM, respectively. This means there are about 109 aptamer molecules per gold particle. Au-DNA Au 0.7 Absorbance 0.6 0.5 0.4 0.3 0.2 0.1 300 400 500 600 700 Wavelength (nm) Figure 6.1. UV-Vis spectra of gold nanoparticles before and after modification with DNA. Two Thrombin aptamers TBA (15-mer) and TBA2 (29-mer) and their analogues were chemically coupled to AuNPs via the formation of Au-S bond. Table 6.1 lists all the DNA-modified AuNPs. AuNPs heavily loaded with linear DNA strands possess strong interparticle electrostatic repulsion, which protects the AuNPs from aggregation in the salt solution (Figure 6.2b). a) b) c) d) Figure 6.2. TEM images of Gold nanoparticles: (a) partially dispersed and aggregated unmodified AuNPs, (b) highly dispersed TBA2-modified AuNPs Au-T6, and aggregation of TBA2-modified AuNPs, Au-T6 in the presence of 10 nM (c) and 20 nM (d) thrombin. 181 6.2.2 Designed Approaches for Detecting Thrombin a) Au Au Target-binding Aptamer (40 nts) Au Aptamer Protein Target + Au b) Au + Protein Target Au Au Au Target-binding Aptamer (40 nts) Figure 6.3. Gold nanoparticle-based colorimetric aptamer biosensors for specifically detecting protein targets. (a) An aptamer/antisense duplex-based biosensors; protein-mediated disruption of DNA duplex disaggregates the nanoparticles, leading to a color change from purple to red detectable by the human eyes, (b) A double aptamer approach that allows the concurrent recognition of two distinct epitopes in the same protein; such binding aggregates the nanoparticals, leading to a color change from red to purple detectable by the human eyes. As illustrated in Figure 6.3a, the DNA aptamer based-duplex biosensor requires three components to be functional: (1) a thiol-modified DNA aptamer of 40-60 nucleotides that can selectively bind to protein target such as human α-thrombin, (2) a thiol-modified shorter complimentary DNA strand containing a region of about 12-20 nucleotides complementary to DNA apamer and (3) gold nanoparticles (13 nm in diameter) functionalized with DNAs that enable the signal read-out by a particle aggregation-dependent change in color. In the absence of protein target, the system is in the aggregated “off” state that appears in purple. This is a result of the complementary between DNAs that leads to the formation of DNA duplexes, which induces the gold nanoparticles to form nanoparticle aggregates. With the addition of protein target, binding between the protein target and protein-binding aptamer may dissociate the duplex, resulting in disassembly of the purple aggregates. Upon 182 disassembly, the color of the system changed from purple (aggregated nanoparticle) or colorless (if aggregated particles precipitate out of solution) to red (individual gold nanoparticle). Such a color change reports and so allows us to “see” the protein in the solution. A double aptamer-based biosensor is described in Figures 6.3b. This approach differs from the duplex approach shown in Figure 6.3a in that two different protein-binding aptamers that have been confirmed to be capable of binding to distinctively different epitopes of the same protein will be used. Before the addition of protein target, gold nanoparticles bearing DNA aptamers of two types will not associate with each other to form aggregates, therefore appearing as red. In the presence of protein target, aptamers will get bound to protein. This brings particles modified with different aptamers into close proximity to form aggregated nanoparticles, which appears as purple. If aggregated particles fully precipitate out of the solution, the solution becomes transparent. Either a color change to purple or observation of precipitate indicates the presence of protein target. This approach shall be highly selective as the chance for other non-target proteins to be recognized simultaneously by two different aptamers is extremely low. 6.2.3 Duplex-based Biosensors for Thrombin Detection Duplex approach (Figure 6.3a) for the detection of human α-thrombin was performed by using two types of DNA-modified AuNPs that carry complementary DNA sequences, that is, Au-T3/Au-T4. In the absence of protein target, the system is 183 in the aggregated state since the complementary between DNAs on Au-T3/Au-T4 leads to the formation of DNA duplexes, inducing the aggregation of gold nanoparticles. According to our design, the addition of thrombin may dissociate the duplex due to the binding between thrombin and thrombin-binding TBA2 in Au-T3, subsequently resulting in disassembly of purple aggregates. However, no color changing could be observed with the addition of thrombin during the experiment. This could be possibly explained by the interaction between complementary DNAs in Au-T3/Au-T4 being stronger than that between thrombin and its aptamer TBA2. 6.2.4 Sensitivity of Double Aptamer Biosensors for Thrombin Detection Au-T5 and Au-T6 are the AuNPs modified with thrombin aptamers TBA and TBA2, respectively, and are designed for the double-aptamer approach. Before the double aptamer biosensor detection is performed, the affinity of each aptamer-based AuNPs toward thrombin was examined. As shown in Figure 6.4, the absorbance peak shifts from 525 nm to 561 nm after the addition of human α-thrombin into Au-T5 solution, indicating the aggregation of AuNPs. As reported, the AuNPs aggregation can occur for Au-T5 in the presence of TBA since TBA can recognize two binding sites on the thrombin, namely, the fibrinogen exosite and the heparin binding site. Besides, steric hindrance should be very minimal since the two binding sites are at opposite ends of thrombin. Therefore, the binding between thrombin and TBA occurs at both sides, causing the clustering of AuNPs. The detection of Au-T5 in our study was 10 nM according to the UV-Vis 184 spectrum, lower than the detection limit of 200 nM previously reported.30 14nM 12nM 10nM 8nM Blank 0.45 0.40 0.35 Absorbance 0.30 0.25 0.20 0.15 0.10 0.05 0.00 -0.05 300 400 500 600 700 800 Wavelength (nm) Figure 6.4. UV-Vis spectra of Au-T5 and that with added thrombin of different concentrations. Surprisingly, both the solution color change and the UV spectrum shift indicate that the aggregation of AuNPs also occurred for Au-T6 with the addition of thrombin into solution (Figures 6.5 and 6.6). Unlike TBA, TBA2 was reported to interact with only one binding site of the thrombin molecule, which is the heparin binding site. We hypothesized that the aggregation of gold nanopraticles may be caused by additional non-specific electrostatic interactions between thrombin and TBA2 after the specific binding between thrombin and TBA2 occurs. Consistent with this, even though the binding affinity of thrombin-binding TBA2 in Au-T6 toward thrombin is almost 50 times higher than TBA in Au-T5, the detection limit by Au-T6 was found to be 30 nM, a value much that by Au-T5 (10 nM). To confirm that the specific binding between thrombin and TBA2 is important for the observed aggregation, Au-T2, containing DNAs incapable of binding thrombin, was studied. No aggregation was observed by adding thrombin at concentrations as high as 250 μM. This result demonstrates that the specific binding site on aptamer TBA2 is very essential for the protein-induced 185 AuNPs aggregation. The aggregation of gold nanoparticles was also monitored by TEM (Figures 6.2c and 6.2d). We observed that the degree of aggregation depends on the thrombin concentration. The same result could be deduced from UV-Vis spectrum Absorbance (Figure. 6.5). 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 300 Au-6+thrombin (40nM) Au-6+thrombin (30nM) Au-6 400 500 600 700 800 Wavelength (nm) Figure 6.5. UV-Vis spectra of Au-T6 with and without thrombin. To provide more evidences that support the importance of specific binding between TBA2 and thrombin in inducing the gold nanoparticle aggregation, DNA molecules that are complementary to TBA2 were added into the solution containing both Au-T6 and thrombin. Our earlier observation and discussion in the duplex approach shows that the interaction of TBA with its complementary DNA strand is stronger than that between TBA and thrombin. If this is also applicable to TBA2, AuNPs should disaggregate since TBA2 forms a more stable duplex structure with its complementary DNAs than that between TBA2 and thrombin, and this turns out to be the case (Figure 6.6c). 186 Figure 6.6. Colorimetric assay based on gold nanoparticles. (a) Au-6 (red), (b) Au-6 with Thrombin (purpule), (c) Au-6 with Thrombin and Apt29R (red). The double aptamer approach was carried out by mixing Au-T5 and Au-T6 in 1:1 ratio. It was found that the detection limit of this method is 10 nM, the same as Au-T5. This result indicates that the sensitivity of double aptamer approach was determined by the weaker binder, e.g., TBA from Au-T5 (Figure 6.7). The influence of both buffers of varying compositions and varying numbers of DNA molecules on the surface of gold nanoparticles on the detection limit was also investigated. Three types of buffers were studied: buffer 1, 50 mM Tris-HCl buffer, pH = 7.5, 140 mM NaCl, mM MgCl, 48 mM KCl, 0.01% Bovine serum albumin (BSA); buffer 2, 10 mM PB buffer, pH = 7.0, 300 mM NaCl; buffer 3, 50 mM Tris-HCl, pH = 8.2, 100 mM NaCl, 1.7 mM CaCl2. It was found that the buffer compositions did not affect the detection limit. By using different concentrations of DNA molecules that were added into the gold colloid solution during the preparation of DNA-modified gold nanoparticles, the number of DNA molecules attached to the gold nanoparticles can be varied. Also, we observed that the number of DNA molecules on Au-NPs (about 60, 80 and 120 DNA molecules per Au-NP) has no detectable influence on the sensitivity by Au-T5 or Au-T6 or the use of both of them 187 for the detection of thrombin. Figure 6.7. Detection limit of gold nanoparticles. (a) Au-T5 (purple), (b) Au-T5/Au-T6 (purple), (c) Au-T6 (red) with the addition of 10 nM thrombin. To examine the effects of the complementary DNAs on the double aptamer approach, either 1μL of Apt15R (a short 15-mer DNA that is complementary to TBA) (100 μM) or Apt29R (a longer 29-mer DNA that is complementary to TBA2) (100 μM) was added into the solution containing Au-T5/Au-T6 (Figure 6.8). The concentration of Apt15R or Apt29R was about times more than that of aptamer on the gold nanoparticles. Considering the self-aggregation of Au-T6 in the presence of thrombin, it is not surprising to see that the aggregation of the gold nanoparticle still proceeded with the addition of Apt15R. Interestingly, the addition of Apt29R inhibited the AuNPs aggregation given that Au-T5 can also self-aggregate in the presence of thrombin since TBAs on the surface of Au-T5 can recognize two thrombin-binding sites. This suggests to us that Au-T5 could only interact with one binding site in thrombin in the presence of excess Apt29R that may occupy the second binding site in thrombin. 188 Figure 6.8. Influence of complementary DNA of aptamers to the detection of thrombin. Addition of thrombin into (a) Au-T5/Au-T6 (left), (b) Au-T5/Au-T6 and Apt29R (middle), (c) Au-T5/Au-T6 and Apt15R (right). 6.2.5 Specificity of Double Aptamer Biosensors To test the specificity of the aptamer-modified AuNPs system for detecting human α-thrombin, several other proteins (human γ-thrombin, bovine α-thrombin, BSA, Factor Xa and lysozyme) were investigated. We chose these proteins according to their sequence similarity to human α-thrombin. There is 85% homology between bovine thrombin and human thrombin, so most antibodies against bovine thrombin cross-react with human thrombin. Rare antibodies against bovine thrombin but not cross-reacting with human thrombin have been reported.37 So human alpha-thrombin has sequence homology of 85% and 37% with bovien alpha-thrombin and factor IX/Xa, respectively. Human γ-thrombin and bovine α-thrombin were firstly studied since bovine α-thrombin shares strong similarity in structure with human α-thrombin and human γ-thrombin is the proteolyzed products of human α-thrombin through enzymatic cleavage or autolytic cleavage. No binding could be detected for human γ-thrombin even at stock solution. However, the detection limit of bovine α-thrombin is 20 μM 189 for Au-T5, 30 nM for Au-T6 and 20 nM for Au-T5/Au-T6, which are almost the same as the human α-thrombin. Proteins other than thrombin for Au-T5/Au-T6 were also examined in Figure 6.9. For BSA, even when the concentration is as higher as mM, no color change was detected. Furthermore, the addition of human α-thrombin (10 nM) into the solution caused a quick color change, indicating the presence of excess BSA did not affect the detection sensitivity of human α-thrombin by Au-NPs. Similarly, Factor Xa at a concentration of μM does not interfere with the detection. Being positively charged at neutral pH and so able to non-specifically interact with negatively charged DNAs on the surface of Au-NP2, lysozyme was found to interfere with the detection when its concentration reaches 0.7 μM, a concentration that is more than 700 times higher than the detection limit of Au-T5/Au-T6. For Au-T5 or Au-T6 alone, the detection limit for those other proteins are the same. To compare the specific interactions between TBA/TBA2 and human α-thrombin and non-specific interactions between TBA/TBA2 and lysozyme, melting temperatures at which Au-NPs disaggregates were determined. We found that the color of AuNPs solution turned from purple to red at 49 oC for human α-thrombin (20 nM), while no color change occurred for lysozyme (20 nM) when the temperature reached as high as 90 oC. This result suggests that the interaction between thrombin and its aptamers attached to the surface of Au-NP2 was not as strong as the non-specific interactions between negatively charged DNAs and positively charged lysozyme at high concentrations. 190 Figure 6.9. Aptamer-modified AuNPs with addition of various proteins at μM. (a) human α-thrombin, (b) BSA, (c) lysozyme, (d) Factor Xa. To investigate the potential application of the aptamer-based AuNPs in a complex biological sample, the interference from the simultaneous presence of many proteins was tested. As expected, a mixture solution of 10-fold excess of human γ-thrombin, BSA and Factor Xa did not interfere with the detection of trace levels (10 nM) of human α-thrombin by Au-T5/T6. 6.3 Conclusion In conclusion, we devised double aptamer approach, a novel gold nanoparticles-based colorimetric method to detect human α-thrombin by taking advantage of two affinity aptamers TBA and TBA2. Compared to duplex approach, this one is more feasible and easy to detect with increased specificity and high sensitivity. This colorimetric method could be applied to other protein detection provided that two different aptamers recognizing the same protein at two distinct epitopes can be found. This may offer an attractive solution to the new generation of 191 advanced portable diagnostic medical devices. 6.4 Experimental Section Preparation of Gold nanoparticles (13nm): AuNPs were prepared by the citrate reduction of HAuCl4. All glassware should be very clean and rinsed with MiliQ H2O. An aqueous solution of HAuCl4 (1 mM, 25 mL) was heat up to 100 oC while stirring, and then 2.5 mL of a 1% trisodium citrate solution was added quickly, which resulted in a change in solution color from pale yellow to deep red. After the color change, the solution was refluxed for an additional 15 min, allowed to cool to room temperature and keep it at RT. The concentration of Au particles is about 13 nM. Preparation of aptamer-modified AuNPs: The 3’- or 5’-terminal disulfide groups of the oligonucleotide strands were first cleaved by soaking them in a 0.1 M dithiothreitol (DTT) phosphate buffer solution (0.1 M phosphate, pH 8.0) for hours and subsequently purified on a NAP-5 column (GE Healthcare). To 800 μL of gold colloid solution was added nmol of the purified oligonucleotide. The solution was brought to 0.3 M NaCl, 10 mM NaH2PO4/Na2HPO4, pH 7.0 buffer (0.3 M PBS) gradually by adding aliquots of M NaCl and 0.1 M NaH2PO4/Na2HPO4, pH 7.0 buffer solutions every 12 hours. After 48 hours, the nanoparticle solutions were centrifuged and redispersed in 750 μL buffer (0.3 M PBS). The final concentrations of the probe solutions were estimated to nM. 192 Reference: 1. Park, S.-J.; Lazarides, A. A.; Mirkin, C. A.; Letsinger, R. L. Angew. Chem. Int. Ed. 2001, 40, 2909. 2. Nam, J.-M.; Park, S.-J.; Mirkin, C. A. J. Am. Chem. 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Recognit. 2003, 16, 23. 194 [...]... containing Au-T5/Au-T6 (Figure 6. 8) The concentration of Apt15R or Apt29R was about 5 times more than that of aptamer on the gold nanoparticles Considering the self-aggregation of Au-T6 in the presence of thrombin, it is not surprising to see that the aggregation of the gold nanoparticle still proceeded with the addition of Apt15R Interestingly, the addition of Apt29R inhibited the AuNPs aggregation given... also self-aggregate in the presence of thrombin since TBAs on the surface of Au-T5 can recognize two thrombin-binding sites This suggests to us that Au-T5 could only interact with one binding site in thrombin in the presence of excess Apt29R that may occupy the second binding site in thrombin 188 Figure 6. 8 Influence of complementary DNA of aptamers to the detection of thrombin Addition of thrombin... the interaction between thrombin and its aptamers attached to the surface of Au-NP2 was not as strong as the non-specific interactions between negatively charged DNAs and positively charged lysozyme at high concentrations 190 Figure 6. 9 Aptamer-modified AuNPs with addition of various proteins at 1 μM (a) human α-thrombin, (b) BSA, (c) lysozyme, (d) Factor Xa To investigate the potential application of. ..Figure 6. 6 Colorimetric assay based on gold nanoparticles (a) Au -6 (red), (b) Au -6 with Thrombin (purpule), (c) Au -6 with Thrombin and Apt29R (red) The double aptamer approach was carried out by mixing Au-T5 and Au-T6 in 1:1 ratio It was found that the detection limit of this method is 10 nM, the same as Au-T5 This result indicates that the sensitivity of double aptamer approach... Au-T6 or the use of both of them 187 for the detection of thrombin Figure 6. 7 Detection limit of gold nanoparticles (a) Au-T5 (purple), (b) Au-T5/Au-T6 (purple), (c) Au-T6 (red) with the addition of 10 nM thrombin To examine the effects of the complementary DNAs on the double aptamer approach, either 1μL of Apt15R (a short 15-mer DNA that is complementary to TBA) (100 μM) or Apt29R (a longer 29-mer DNA... into (a) Au-T5/Au-T6 (left), (b) Au-T5/Au-T6 and Apt29R (middle), (c) Au-T5/Au-T6 and Apt15R (right) 6. 2.5 Specificity of Double Aptamer Biosensors To test the specificity of the aptamer-modified AuNPs system for detecting human α-thrombin, several other proteins (human γ-thrombin, bovine α-thrombin, BSA, Factor Xa and lysozyme) were investigated We chose these proteins according to their sequence similarity... specific interactions between TBA/TBA2 and human α-thrombin and non-specific interactions between TBA/TBA2 and lysozyme, melting temperatures at which Au-NPs disaggregates were determined We found that the color of AuNPs solution turned from purple to red at 49 oC for human α-thrombin (20 nM), while no color change occurred for lysozyme (20 nM) when the temperature reached as high as 90 oC This result suggests... approach was determined by the weaker binder, e .g. , TBA from Au-T5 (Figure 6. 7) The influence of both buffers of varying compositions and varying numbers of DNA molecules on the surface of gold nanoparticles on the detection limit was also investigated Three types of buffers were studied: buffer 1, 50 mM Tris-HCl buffer, pH = 7.5, 140 mM NaCl, 1 mM MgCl, 48 mM KCl, 0.01% Bovine serum albumin (BSA);... There is 85% homology between bovine thrombin and human thrombin, so most antibodies against bovine thrombin cross-react with human thrombin Rare antibodies against bovine thrombin but not cross-reacting with human thrombin have been reported.37 So human alpha-thrombin has sequence homology of 85% and 37% with bovien alpha-thrombin and factor IX/Xa, respectively Human γ-thrombin and bovine α-thrombin... bovine α-thrombin shares strong similarity in structure with human α-thrombin and human γ-thrombin is the proteolyzed products of human α-thrombin through enzymatic cleavage or autolytic cleavage No binding could be detected for human γ-thrombin even at stock solution However, the detection limit of bovine α-thrombin is 20 μM 189 for Au-T5, 30 nM for Au-T6 and 20 nM for Au-T5/Au-T6, which are almost the . immobilized on gold nanoparticle Au-T5 Au-SH-3’-AAAAAAAAAACTA GGTTGGTGT GGTTGG-5’ TBA1 immobilized on gold nanoparticle Au-T6 Au-SH-3’-AAAAAAAAAACTA TCAGTGGGGTTGGACGGGATGGTGCCTGA-5’ TBA2 immobilized. (5’-GGTTGGTGTGGTTGG-3’), which binds to the fibrinogen-binding site of thrombin with Kd ≈ 100 nM, and 29-mer TBA2 (5’-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3’), which binds to the heparin-binding site of thrombin. Au-SH-3’-AAAAAAAAAACTATCAGTGGGGTTGGACGGGATGGTGCCTG AT GTAT C- 5’ Modified TBA2 immobilized on gold nanoparticle Au-T4 Au-SH-3’-AAAAAAAAAACTA GATACA TCAGGC-5’ Complementary sequence of modified TBA1