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==1st Trial==
==1st Trial==


 
<h4>1. Conjugation condition</h4>
 
<b>Conjugation condition</b>
 
Single strand DNA, A2 and A3 conjugated with chlorambrucil and folic acid respectively in ratio 1:1.
Single strand DNA, A2 and A3 conjugated with chlorambrucil and folic acid respectively in ratio 1:1.
Mixtures were vortexed at room temperature for 4 hrs.
Mixtures were vortexed at room temperature for 4 hrs.


In-Complete route
In-Complete route
Folic acid and chlorambrucil were first conjugated and folded, carbon nanodots were added afterwards at ration 1:1.
Folic acid and chlorambrucil were first conjugated and folded, carbon nanodots were added afterwards at ration 1:1.




<h4>Mass-spectrometry</h4>
<h4>2. Mass-spectrometry</h4>
1. Control group 1: Single-stranded DNA A2<br>
i. Control Group:<br>
 
[[Image:Masssp2.jpg|720px|thumb|alt=Example alt text|Fig.1(A)Control group one: MS-Single stranded DNA A2<br>(B)Control group two: MS-Single stranded DNA A3<br>]]
[[Image:Mass_Spectrometry_Control_Single-stranded_DNA_A2.png|500px|For MS-single stranded DNA A2 control ]]
ii. Conjugate Group:<br>
 
[[Image:Exp1.2.jpg|720px|thumb|alt=Example alt text|Fig.2(A)For MS-single stranded DNA A2 ,a minor peak at 10120 shows the conjugation of A2 and chlorambrucil.<br>(B)For MS-single stranded DNA A3 , a minor peak at 7043 shows the conjugation of A3 and folic acid.<br>(C)For MS-single stranded DNA A6 , a peak at 11171 shows the conjugation of A6 and carbon nanodots<br>]]
2. Control group 2: Single-stranded DNA A3<br>
 
[[Image:Mass_spectrometry_Single-stranded_DNA_A3_.png‎|500px|For MS-single stranded DNA A3 control ]]
 
 
3. Conjugated group<br>
 
[[Image:Exp1.2.jpg|720px|For MS-single stranded DNA A2 , a minor peak at 10120 shows the conjugation of A2 and chlorambrucil.<br>
 
For MS-single stranded DNA A3 , a minor peak at 7043 shows the conjugation of A3 and folic acid.<br>
 
For MS-single stranded DNA A6 , a peak at 11171 shows the conjugation of A6 and carbon nanodots. ]]
 
For MS-single stranded DNA A2 ,a minor peak at 10120 shows the conjugation of A2 and chlorambrucil.<br>
 
For MS-single stranded DNA A3 , a minor peak at 7043 shows the conjugation of A3 and folic acid.<br>
 
For MS-single stranded DNA A6 , a peak at 11171 shows the conjugation of A6 and carbon nanodots<br>
 
<h4>DNA extraction after folding of DNA origami</h4>
Gel photo :


[[Image:expresultSection1_4.jpg|400px|
Left: GeneRuler DNA Ladders, Samples, Modified Primers A2, A3 ]] <br>


<h4>Nanodrop Reading</h4>
<h4>3. DNA extraction after folding of DNA origami</h4>
DNA concentration : 0.8 ng/ul
Gel photo :<br>
[[Image:expresultSection1_4.jpg|400px|]] <br>
(Fig.3, GeneRuler DNA Ladders, Samples, Modified Primers A2, A3) <br>


260/280 : 1.34


<h4>4. Nanodrop Reading</h4>
DNA concentration : <br>
0.8 ng/ul<br>
260/280 : 1.34<br>
260/230 : 0.86
260/230 : 0.86




 
<h4>5. Confocal Microscopy</h4>
 
[[Image:C1.jpg|720px|thumb|alt=Example alt text|Fig.4(A-C)Hep G2 Treatment ; (D-F)Hep G2 Control<br>
<h4>Confocal Microscopy</h4>
(Excitation wave lengths: 405nm, Emission wave lengths: 460 – 560nm)<br>
Hep G2 Treatment
The intensity for the fluorescence is very low. It is interpreted that a minimal amount of drug has entered the cells which may due to low concentration of drug in condition of 1st trial.]]<br><br><br>
 
[[Image:expresultSection1_5.jpg|230px]]  [[Image:expresultSection1_6.jpg|230px]]  [[Image:expresultSection1_7.jpg|230px]]
 
 
Hep G2 Control
 
[[Image:expresultSection1_8.jpg|230px]]  [[Image:expresultSection1_9.jpg|230px]]  [[Image:expresultSection1_10.jpg|230px]]
 
Excitation wave lengths: 405nm
 
Emission wave lengths: 460 – 560nm


==2nd Trial==
==2nd Trial==


<b>Conjugation condition</b>
<h4>1. Conjugation condition</h4>
 
Single strand DNA, A2 and A3 conjugated with chlorambrucil and folic acid respectively in ratio 1:1. Mixtures were vortexed at room temperature for 4 hrs
Single strand DNA, A2 and A3 conjugated with chlorambrucil and folic acid respectively in ratio 1:1. Mixtures were vortexed at room temperature for 4 hrs.
<br>
 
 
In-Complete route
In-Complete route
Folic acid and chlorambrucil were first conjugated and folded, carbon nanodots were added afterwards at ration 1:1.  
Folic acid and chlorambrucil were first conjugated and folded, carbon nanodots were added afterwards at ration 1:1.  




<h4>Mass-spectrometry</h4>
<h4>2. Mass-spectrometry</h4>
 
i. Control Group 1 & 2 refer to Trial 1 Fig.1<br>
1. Control group 1 & 2 refer to Trial 1<br>
ii. Conjugated Group:<br>
 
[[Image:Exp2.jpg|720px|thumb|alt=Example alt text|Fig.5(A)For MS-single stranded DNA A2 , a minor peak at 10120 shows the conjugation of A2 and chlorambrucil.<br>(B)For MS-single stranded DNA A3 , a minor peak at 7043 shows the conjugation of A3 and folic acid.]]
2. Conjugated group<br>
<br>
 
<br>
[[Image:Exp2.jpg|720px|
For MS-single stranded DNA A2 , a minor peak at 10120 shows the conjugation of A2 and chlorambrucil.
 
For MS-single stranded DNA A3 , a minor peak at 7043 shows the conjugation of A3 and folic acid. ]]


For MS-single stranded DNA A2 , a minor peak at 10120 shows the conjugation of A2 and chlorambrucil.<br>
<h4>3. DNA extraction after folding of DNA origami</h4>
Gel photo :<br>
[[Image:expresultSection2_3.jpg|400px|]]<br>
(Fig.6, GeneRuler DNA Ladders, Samples, Modified Primers A2, A3)<br>  


For MS-single stranded DNA A3 , a minor peak at 7043 shows the conjugation of A3 and folic acid.<br>


<h4>DNA extraction after folding of DNA origami</h4>
<h4>4. Nanodrop Reading</h4>
DNA concentration :<br>
2.3 ng/ul<br>
260/280 : 1.28<br>
260/230 : 0.52<br>


Gel photo :
[[Image:expresultSection2_3.jpg|400px|
Left: GeneRuler DNA Ladders, Samples, Modified Primers A2, A3]]


 
<h4>5. Confocal Microscopy</h4>
<h4>Nanodrop Reading</h4>
[[Image:C2.jpg|720px|thumb|alt=Example alt text|Fig.7(A-C)Hep G2 Treatment ; (D-F)Hep G2 Control<br>
 
(Excitation wave lengths: 405nm, Emission wave lengths: 460 – 560nm)<br>
DNA concentration : 2.3 ng/ul
The intensity for the fluorescence is enhanced with respect to 1st Trial. More drugs were entering the cell this time.]]
 
260/280 : 1.28
 
260/230 : 0.52
 
<h4>Confocal Microscopy</h4>
Hep G2 Treatment
 
[[Image:expresultSection2_4.jpg|230px]]  [[Image:expresultSection2_5.jpg|230px]]  [[Image:expresultSection2_6.jpg|230px]]
 
 
Hep G2 Control
 
[[Image:expresultSection2_7.jpg|230px]]  [[Image:expresultSection2_8.jpg|230px]]  [[Image:expresultSection2_9.jpg|230px]]
 
Excitation wave lengths: 405nm
 
Emission wave lengths: 460 – 560nm


==3rd Trial==
==3rd Trial==


<b>Conjugation condition</b>
<h4>1. Conjugation condition</h4>
 
 
Single strand DNA, A2, A3 and A6 conjugated with chlorambrucil, folic acid and carbon nanodots respectively in ratio 1:10.
Single strand DNA, A2, A3 and A6 conjugated with chlorambrucil, folic acid and carbon nanodots respectively in ratio 1:10.
Mixtures were vortexed at 37 for 4 hrs.
Mixtures were vortexed at 37 for 4 hrs.
 
Complete route<br>
 
 
Complete route
 
All substituent was conjugated and folded simultaneously
All substituent was conjugated and folded simultaneously




<h4>Mass-spectrometry</h4>
<h4>2. Mass-spectrometry</h4>
1. Control group 1 & 2 refer to Trial 1<br>
i. Control group 1 & 2 refer to Trial 1 Fig.1<br>
ii. Conjugated group<br>
[[Image:Exp3.jpg|720px|thumb|alt=Example alt text|Fig.8(A)For MS-single stranded DNA A2 , a peak at 10193 shows the conjugation of A2 and chlorambrucil.<br>(B)For MS-single stranded DNA A3 , a peak at 7043 shows the conjugation of A3 and folic acid.<br>]]


2. Conjugated group<br>


[[Image:Exp3.jpg|720px|thumb|alt=Example alt text|For MS-single stranded DNA A2 , a peak at 10193 shows the conjugation of A2 and chlorambrucil.<br>For MS-single stranded DNA A3 , a peak at 7043 shows the conjugation of A3 and folic acid.<br>]]
<h4>3. DNA extraction after folding of DNA origami</h4>
Gel photo :<br>
[[Image:expresultSection3_3.jpg|400px]]<br>
(Fig.9, GeneRuler DNA Ladders, Samples)<br>




<h4>4. Nanodrop Reading</h4>
DNA concentration : <br>5.5 ng/ul<br>
260/280 : 2.65<br>
260/230 : 1.12<br>


<h4>DNA extraction after folding of DNA origami</h4>


Gel photo :
<h4>5. Confocal Microscopy</h4>
[[Image:C3.jpg|720px|thumb|alt=Example alt text|<br>Fig.10(A-C)Hep G2 Treatment ; (D-F)Hep G2 Control<br>
(Excitation : 405nm, Emission : 460 – 560nm)<br>
The intensity for the fluorescence is further enhanced with respect to 1st and 2nd Trial. The signal peaks at 473nm. A lot of drugs were entering the cell. It may due to the enhancement done to the reaction condition of drugs.]]
[[Image:C4.jpg|720px|thumb|alt=Example alt text|<br>Fig.11(A-C)HK-1 Treatment ; (D-F)HK-1 Control<br>
(Excitation : 405nm, Emission : 460 – 560nm)]]


[[Image:expresultSection3_3.jpg|400px]]


<h4>6. MTT assay</h4>
[[Image:MTT_assay.png|527px]]<br>
Fig.12: 16 samples were used for the MTT Assay and HK-1 was the target cancer cell.<br>
It is found that 12.34% of cells is killed after treatment.<br>
The standard deviation for the result is 0.01259.<br>


<h4>Nanodrop Reading</h4>


DNA concentration : 5.5 ng/ul
<h4>7. Emission Spectrum</h4>
i. Hep G2:<br>                                
[[Image:expresultSection3_16.jpg|300px]] <br><br>
ii. HK-1:<br>
[[Image:expresultSection3_17.jpg|300px]]


260/280 : 2.65


260/230 : 1.12
<h4>8. AFM photo</h4>
[[Image:expresultSection3_18.jpg|300px]]  [[Image:expresultSection3_19.jpg|300px]]


[[Image:expresultSection3_20.jpg|300px]]  [[Image:expresultSection3_21.jpg|300px]]


<h4>Confocal Microscopy</h4>
==Discussion==
Hep G2 Treatment


[[Image:expresultSection3_4.jpg|230px]]  [[Image:expresultSection3_5.jpg|230px]]  [[Image:expresultSection3_6.jpg|230px]]
<b>1. MAILDI-TOF mass spectrometry</b><br>
MALDI-TOF mass spectrometry was used to measure the reaction yield of the conjugation between single strand DNA and chlorambucil,<br> FOLIC ACID or graphite quantum dots. A relatively low yield was found according to the mass spectrum.<br>


In order to obtain a higher product yield, we raised the reaction temperature little by little and attempted 0oC, 25oC, 37oC and 50oC. It is obvious that with the increase of reaction temperature, the product yield also enhances, which can be seen from the mass spectrums. On the other hand, we still raised the reactant ratio between the single strand DNA and molecules from 1:1 to 1:10. However, very little improvement was observed in the yield. That may be because both chlorambucil and FOLIC ACID have a very low solubility in water. So an increase in reactant ratio has less influence on the product yield than in reaction temperature.Here, we present the data under two kinds of reaction condition.<br>


Hep G2 Control
a.The single strand DNA was conjugated with chlorambucil, FOLIC ACID or GQD with 1:1 ratio. And the reaction was undergone at the room temperature about 25 oC.<br>
b.The single strand DNA was conjugated with chlorambucil, folic acid or GQD with 1:10 ratio. And the reaction was undergone at 37 oC.<br>


[[Image:expresultSection3_7.jpg|230px]]  [[Image:expresultSection3_8.jpg|230px]]  [[Image:expresultSection3_9.jpg|230px]]
<b>2. Atomic force spectrometry</b><br>
 
The Atomic force microscopy image has a low resolution and the structure of the functionalized DNA origami
HK-1 Treatment
can hardly be identified which may due to the following reasons:<br>
 
[[Image:expresultSection3_10.jpg|230px]]  [[Image:expresultSection3_11.jpg|230px]]  [[Image:expresultSection3_12.jpg|230px]]
 
 
HK-1 Control
 
[[Image:expresultSection3_13.jpg|230px]]  [[Image:expresultSection3_14.jpg|230px]]  [[Image:expresultSection3_15.jpg|230px]]
 
Excitation : 405nm
 
Emission : 460 – 560nm
 
 
<h4>Emission Spectrum</h4>
Hep G2                                           HK-1
 
[[Image:expresultSection3_16.jpg|300px]]  [[Image:expresultSection3_17.jpg|300px]]
 
<h4>AFM photo</h4>
[[Image:expresultSection3_18.jpg|300px]]  [[Image:expresultSection3_19.jpg|300px]]


[[Image:expresultSection3_20.jpg|300px]]  [[Image:expresultSection3_21.jpg|300px]]
a.The DNA origami samples is not dried up completely which increase the ADHESIVE force of the AFM instrument<br>
b.The DNA origami samples was clustered on the mica platform<br>
c.The scan size of AFM is restricted from 1 to 5 nm so many area attached with the DNA was not examined properly.<br>


<b>Discussion:</b><br>
==Future Development==
The Atomic force microscopy image has a low resolution and the structure of the functionalized DNA origami<br>
1. Modify the DNA origami design with more surface area to accommodate more folic acid, chlorambucil and graphene quantum dot<br>
can hardly be identified which may due to the following reasons:<br>
2. Increase the drug load to enhance the cytotoxicity of the functionalized DNA origami complex<br>
# The DNA origami samples is not dried up completely which increase the adhesive force of the AFM instrument<br>
3. Adapt other specific biomarker from cancer cell to increase the targeting efficiency<br>
# The DNA origami samples was clustered on the mica platform<br>
# The scan size of AFM is restricted from 1 to 5 nm so many area attached with the DNA was not examined properly<br>

Latest revision as of 21:19, 25 October 2014

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1st Trial

1. Conjugation condition

Single strand DNA, A2 and A3 conjugated with chlorambrucil and folic acid respectively in ratio 1:1. Mixtures were vortexed at room temperature for 4 hrs.

In-Complete route Folic acid and chlorambrucil were first conjugated and folded, carbon nanodots were added afterwards at ration 1:1.


2. Mass-spectrometry

i. Control Group:

Example alt text
Fig.1(A)Control group one: MS-Single stranded DNA A2
(B)Control group two: MS-Single stranded DNA A3

ii. Conjugate Group:

Example alt text
Fig.2(A)For MS-single stranded DNA A2 ,a minor peak at 10120 shows the conjugation of A2 and chlorambrucil.
(B)For MS-single stranded DNA A3 , a minor peak at 7043 shows the conjugation of A3 and folic acid.
(C)For MS-single stranded DNA A6 , a peak at 11171 shows the conjugation of A6 and carbon nanodots


3. DNA extraction after folding of DNA origami

Gel photo :

(Fig.3, GeneRuler DNA Ladders, Samples, Modified Primers A2, A3)


4. Nanodrop Reading

DNA concentration :
0.8 ng/ul
260/280 : 1.34
260/230 : 0.86


5. Confocal Microscopy

Example alt text
Fig.4(A-C)Hep G2 Treatment ; (D-F)Hep G2 Control
(Excitation wave lengths: 405nm, Emission wave lengths: 460 – 560nm)
The intensity for the fluorescence is very low. It is interpreted that a minimal amount of drug has entered the cells which may due to low concentration of drug in condition of 1st trial.



2nd Trial

1. Conjugation condition

Single strand DNA, A2 and A3 conjugated with chlorambrucil and folic acid respectively in ratio 1:1. Mixtures were vortexed at room temperature for 4 hrs
In-Complete route Folic acid and chlorambrucil were first conjugated and folded, carbon nanodots were added afterwards at ration 1:1.


2. Mass-spectrometry

i. Control Group 1 & 2 refer to Trial 1 Fig.1
ii. Conjugated Group:

Example alt text
Fig.5(A)For MS-single stranded DNA A2 , a minor peak at 10120 shows the conjugation of A2 and chlorambrucil.
(B)For MS-single stranded DNA A3 , a minor peak at 7043 shows the conjugation of A3 and folic acid.



3. DNA extraction after folding of DNA origami

Gel photo :

(Fig.6, GeneRuler DNA Ladders, Samples, Modified Primers A2, A3)


4. Nanodrop Reading

DNA concentration :
2.3 ng/ul
260/280 : 1.28
260/230 : 0.52


5. Confocal Microscopy

Example alt text
Fig.7(A-C)Hep G2 Treatment ; (D-F)Hep G2 Control
(Excitation wave lengths: 405nm, Emission wave lengths: 460 – 560nm)
The intensity for the fluorescence is enhanced with respect to 1st Trial. More drugs were entering the cell this time.

3rd Trial

1. Conjugation condition

Single strand DNA, A2, A3 and A6 conjugated with chlorambrucil, folic acid and carbon nanodots respectively in ratio 1:10. Mixtures were vortexed at 37 for 4 hrs. Complete route
All substituent was conjugated and folded simultaneously


2. Mass-spectrometry

i. Control group 1 & 2 refer to Trial 1 Fig.1
ii. Conjugated group

Example alt text
Fig.8(A)For MS-single stranded DNA A2 , a peak at 10193 shows the conjugation of A2 and chlorambrucil.
(B)For MS-single stranded DNA A3 , a peak at 7043 shows the conjugation of A3 and folic acid.


3. DNA extraction after folding of DNA origami

Gel photo :

(Fig.9, GeneRuler DNA Ladders, Samples)


4. Nanodrop Reading

DNA concentration :
5.5 ng/ul
260/280 : 2.65
260/230 : 1.12


5. Confocal Microscopy

Example alt text

Fig.10(A-C)Hep G2 Treatment ; (D-F)Hep G2 Control
(Excitation : 405nm, Emission : 460 – 560nm)
The intensity for the fluorescence is further enhanced with respect to 1st and 2nd Trial. The signal peaks at 473nm. A lot of drugs were entering the cell. It may due to the enhancement done to the reaction condition of drugs.
Example alt text

Fig.11(A-C)HK-1 Treatment ; (D-F)HK-1 Control
(Excitation : 405nm, Emission : 460 – 560nm)


6. MTT assay


Fig.12: 16 samples were used for the MTT Assay and HK-1 was the target cancer cell.
It is found that 12.34% of cells is killed after treatment.
The standard deviation for the result is 0.01259.


7. Emission Spectrum

i. Hep G2:


ii. HK-1:


8. AFM photo

Discussion

1. MAILDI-TOF mass spectrometry
MALDI-TOF mass spectrometry was used to measure the reaction yield of the conjugation between single strand DNA and chlorambucil,
FOLIC ACID or graphite quantum dots. A relatively low yield was found according to the mass spectrum.

In order to obtain a higher product yield, we raised the reaction temperature little by little and attempted 0oC, 25oC, 37oC and 50oC. It is obvious that with the increase of reaction temperature, the product yield also enhances, which can be seen from the mass spectrums. On the other hand, we still raised the reactant ratio between the single strand DNA and molecules from 1:1 to 1:10. However, very little improvement was observed in the yield. That may be because both chlorambucil and FOLIC ACID have a very low solubility in water. So an increase in reactant ratio has less influence on the product yield than in reaction temperature.Here, we present the data under two kinds of reaction condition.

a.The single strand DNA was conjugated with chlorambucil, FOLIC ACID or GQD with 1:1 ratio. And the reaction was undergone at the room temperature about 25 oC.
b.The single strand DNA was conjugated with chlorambucil, folic acid or GQD with 1:10 ratio. And the reaction was undergone at 37 oC.

2. Atomic force spectrometry
The Atomic force microscopy image has a low resolution and the structure of the functionalized DNA origami can hardly be identified which may due to the following reasons:

a.The DNA origami samples is not dried up completely which increase the ADHESIVE force of the AFM instrument
b.The DNA origami samples was clustered on the mica platform
c.The scan size of AFM is restricted from 1 to 5 nm so many area attached with the DNA was not examined properly.

Future Development

1. Modify the DNA origami design with more surface area to accommodate more folic acid, chlorambucil and graphene quantum dot
2. Increase the drug load to enhance the cytotoxicity of the functionalized DNA origami complex
3. Adapt other specific biomarker from cancer cell to increase the targeting efficiency

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