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

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,

Chapter 6

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,

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

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

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

(5’-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3’), which binds to the

heparin-binding site of thrombin with Kd ≈ 0.5 nM) 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 2

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,

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

6.2 Result and Discussion

6.2.1 Preparation of Aptamer-based Gold Nanoparticles

Table 6.1 DNA and DNA-modified AuNPs

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

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-SH-3’-AAAAAAAAAACTATCAGTGGGGTTGGACGGGATGGTGCCTG

ATGTATC-5’

Modified TBA2 immobilized on gold nanoparticle

Au-T4 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

TCAGTGGGGTTGGACGGGATGGTGCCTGA-5’

TBA2 immobilized on gold nanoparticle

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

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

0.1 0.2 0.3 0.4 0.5 0.6 0.7

Wavelength (nm)

Au-DNA Au

6.2.2 Designed Approaches for Detecting Thrombin

a)

b)

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

Au

Au Protein Target

Au

Au

Aptamer

+

Target-binding Aptamer (40 nts)

Au

Au

Protein Target

Target-binding Aptamer (40 nts)

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

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

spectrum, lower than the detection limit of 200 nM previously reported.30

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

-0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45

Wavelength (nm)

14nM 12nM 10nM 8nM Blank

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

(Figure 6.5)

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)

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70

Wavelength (nm)

Au-6+thrombin (40nM) Au-6+thrombin (30nM) Au-6