Plan de beneficios definidos-

The activity of enzyme is significantly different between surface-phase reaction and solution-solution reaction. The hybridization of the target on AuNP-conjugated DNA probe is possibly inhibited by non-specific adsorption of DNA on the surface, unfavorable confirmation of DNA probe on the surface and limited space for DNA binding on the gold nanoparticle surface. To solve these problems, we used a thiolated small molecule dithiothreitol (DTT) as back-filler to remove non-specific DNA and reduce DNA surface coverage on the surface of the gold nanoparticle, facilitating favorable DNA hybridization on the nanoparticle surface. We further investigated the kinetics of the enzyme using the DTT-treated AuNPs.

5.3.4.1. DTT as a back-filler

Alkanethiols such as mercaptohexanol (MCH), mercaptoethanol (MCE) and DTT (dithiothreitol) have been widely used to immobilize on the gold surface to form self- assembly monolayer (SAM) to lift DNA probe for enhanced DNA hybridization

efficiency.262,263 This backfilling of alkanethiols ensures the vertical orientation of the

thiolated DNA probe and also reduces non-specific DNA adsorption and DNA surface coverage. It has been proven that the back-filling of DTT on the surface of the gold

electrode is better than that of MCH.264 Although such back-filling of the alkanethiols is

straight forward on electrode surfaces, it is somehow challenged for the surface of AuNPs due to the poor stability of gold nanoparticle at low DNA surface coverage.

Recently, Zhao et al.161 have used a low concentration of MCH as a back-filler to

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Figure 44. DTT as a back-filler for controlling DNA surface coverage on the DNA-

modified AuNPs.

In this study, we used DTT as new back filler to control DNA surface coverage on AuNP surface (Figure 44). DTT is a strong reducing agent with a redox potential of -

0.33V at pH 7.265 The reduction of disulfide bond proceeds by two subsequent thiol-

disulphide exchange reactions, resulting in the cyclic structure on the surface of the AuNPs. A very high concentration of DTT, typically in molar range, has been used to

remove DNA probe from the AuNP151 by ligand displacement reaction due to its higher

affinity to the gold surface as compared to the thiol modified DNA. To partially remove the probe DNA on from the modified AuNP, we have used very low concentration of DTT in µM range to control DNA surface coverage on the surface of the gold nanoparticle. We hypothesize that it works as a backfiller to change the horizontal or tilted orientation of DNA strands attached to gold nanoparticle into the vertical ones. During the reaction, it may also remove most non-specifically adsorb and some specifically adsorbed DNA from the surface.

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5.3.4.2. Kinetics of DTT-displacement for DNA-modified AuNPs

To find out whether DTT reduced the DNA surface coverage on the surface of the modified AuNPs, we carried out fluorescence experiments using the AuNP-conjugated FAM-labeled DNA probe. Figure 45 represents the kinetics of the FAM-labeled probe modified AuNPs treated with different concentrations of DTT. Although fluorescence of the FAM probe is quenched by AuNPs, we were able to monitor the fluorescence change of the modified AuNPs with different concentration of DTT.

0 10 20 30 15000 30000 45000 50 mM 20 M 10 M 5 M Flu or escen ce (a .u.) Time (minutes) 0 M

Figure 45. Kinetics of displacement of FAM-labeled DNA from the surface of the gold

nanoparticle by DTT.

In the absence of DTT, there was small fluorescence change of about 3000 a.u. during the measurement period, indicating the addition of buffer might have small effect on FAM fluorescence. However, with the addition of DTT solution from 5 µM to 50 µM, the fluorescence of the solution increased, indicating that DTT either changed orientation or/and removed FAM-labeled DNA from the AuNP surface. Both the change in orientation and release of FAM-labeled DNA into the solution increased the fluorescence due to a decreased quenching efficiency of AuNPs.

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5.3.4.3 Effect of DTT treatment on surface coverage of the DNA-modified AuNPs

60-DTT 50-DTT 35-DTT 20-DTT 10-DTT 0-DTT 0 20 40 60 80 100 Cover age (Str and s/pa rt icle) 0 70 140 210 280 0 15000 30000 45000 Flu or escen ce (a .u.) Concentration (nM) Samples

Figure 46. Effect of DTT treatment on the surface coverage of DNA on the surface of

AuNP. (A) Calibration curve to determine the change in surface coverage with different concentration of DTT and (B) change in surface coverage as a function of DTT treatment.

To find out the change of DNA surface coverage after the treatment with low concentration of DTT, we separated the DTT-treated AuNPs by washing and centrifugation as described in the procedure. The DNA surface coverage of DTT-treated AuNPs was determined by completely removing DNA from the surface of the gold nanoparticle using the high concentration of DTT (0.5M). Figure 46A represents a calibration curve of standard 1X_FAM probe solutions in the range of 0 to 250 nM with

linear regression equation of 𝑌 = 179.04 𝐶 (𝑛𝑀) − 298.98 (𝑟 = 0.9994). Using this

established calibration curve, we calculated the DNA surface coverage of different DTT- treated samples under same conditions (Figure 46B). The surface coverage of 0, 20, 35, 50, 60 μM DTT-treated samples was found to be 86, 80, 77, 68, 60, 59 DNA strand per

AuNP which is equivalent to 26.9, 25.0, 24.0, 21.2, 18.7 and 18.4 pmole/cm2,

respectively. Clearly, the DNA surface coverage decreased from its initial value (86 DNA strand per AuNP) to 59 DNA strand per AuNP with 60 µM DTT treatment for 30 minutes.

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5.3.5. Activity of Exonuclease III on DTT-treated DNA-modified AuNPs