Team:Calgary/Project/OurSensor/Reporter/PrussianBlueFerritin

From 2013.igem.org

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<p><b>Figure 1.</b> Ribbon visualization of a fully assembled ferritin protein.</p>
<p><b>Figure 1.</b> Ribbon visualization of a fully assembled ferritin protein.</p>
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<p><b>Figure 2.</b> Chemical equation for the disproportionation of hydrogen peroxide into oxygen radical species using iron as a catalyst; Fenton chemistry. </p>
<p><b>Figure 2.</b> Chemical equation for the disproportionation of hydrogen peroxide into oxygen radical species using iron as a catalyst; Fenton chemistry. </p>
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<p><b>Figure 3.</b> Chemical equation for the synthesis of Prussian blue ferritin using potassium ferrocyanide.</p>
<p><b>Figure 3.</b> Chemical equation for the synthesis of Prussian blue ferritin using potassium ferrocyanide.</p>
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<p><b>Figure 4.</b> Comparison image of commercial ferritin to Prussian blue ferritin after the synthesis reaction. The synthesis reaction took place over a 12 hour time period. </p>
<p><b>Figure 4.</b> Comparison image of commercial ferritin to Prussian blue ferritin after the synthesis reaction. The synthesis reaction took place over a 12 hour time period. </p>
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<p><b>Figure 5.</b> Measurements of the absorbance of the 650nm light by the substrate TMB over a period of 600 seconds. 6 uL of 10 mg/mL substrate was used in a 242 uL reaction volume.Commercial Prussian blue ferritin ( 10 uL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 uL of 0.047 mg/mL sample). Negative controls are TMB and hydrogen peroxide, and TMB only. Standard error of the mean bars are based on a sample size where n=8. Substrate and hydrogen peroxide sample data is not clearly visible as it is in line with the substrate only data. </p>
<p><b>Figure 5.</b> Measurements of the absorbance of the 650nm light by the substrate TMB over a period of 600 seconds. 6 uL of 10 mg/mL substrate was used in a 242 uL reaction volume.Commercial Prussian blue ferritin ( 10 uL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 uL of 0.047 mg/mL sample). Negative controls are TMB and hydrogen peroxide, and TMB only. Standard error of the mean bars are based on a sample size where n=8. Substrate and hydrogen peroxide sample data is not clearly visible as it is in line with the substrate only data. </p>
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<p><b>Figure 6.</b> Measurements of the absorbance of the 415nm light by the substrate ABTS over a period of 600 seconds. 8 uL of 10 mg/mL substrate was used in a 242 uL reaction volume. Commercial Prussian blue ferritin ( 10 uL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 uL of 0.047 mg/mL sample). Negative controls are ABTS and hydrogen peroxide, and ABTS only. Standard error of the mean bars are based on a sample size where n=8.</p>
<p><b>Figure 6.</b> Measurements of the absorbance of the 415nm light by the substrate ABTS over a period of 600 seconds. 8 uL of 10 mg/mL substrate was used in a 242 uL reaction volume. Commercial Prussian blue ferritin ( 10 uL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 uL of 0.047 mg/mL sample). Negative controls are ABTS and hydrogen peroxide, and ABTS only. Standard error of the mean bars are based on a sample size where n=8.</p>
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<p><b>Figure 7.</b> pH optimization of commercial Prussian blue ferritin with ABTS. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 415 nm to detect the colourimetric change in a 242 uL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility. </p>
<p><b>Figure 7.</b> pH optimization of commercial Prussian blue ferritin with ABTS. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 415 nm to detect the colourimetric change in a 242 uL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility. </p>
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<p><b>Figure 8.</b> pH optimization of commercial Prussian blue ferritin with TMB. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 650 nm to detect the colourimetric change in a 242 uL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility. </p>
<p><b>Figure 8.</b> pH optimization of commercial Prussian blue ferritin with TMB. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 650 nm to detect the colourimetric change in a 242 uL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility. </p>
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<p><b>Figure 9.</b> Temperature optimization of commercial Prussian blue ferritin with ABTS. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 415 nm to detect the colourimetric change in a 242 uL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility. </p>
<p><b>Figure 9.</b> Temperature optimization of commercial Prussian blue ferritin with ABTS. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 415 nm to detect the colourimetric change in a 242 uL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility. </p>
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<p><b>Figure 10.</b> Temperature optimization of commerical Prussian blue ferritin with TMB. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 650 nm to detect the colourimetric change in a 242 uL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility. </p>
<p><b>Figure 10.</b> Temperature optimization of commerical Prussian blue ferritin with TMB. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 650 nm to detect the colourimetric change in a 242 uL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility. </p>
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<p><b>Figure 11.</b> Image of the colours of ABTS and TMB (10 mg/mL for both) after reacting with Prussian blue ferritin.</p>
<p><b>Figure 11.</b> Image of the colours of ABTS and TMB (10 mg/mL for both) after reacting with Prussian blue ferritin.</p>
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<p><b>Figure 12.</b> Blots of Prussian blue ferritin on nitrocellulose (20 uL samples) that are reacted with ABTS (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.</p>
<p><b>Figure 12.</b> Blots of Prussian blue ferritin on nitrocellulose (20 uL samples) that are reacted with ABTS (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.</p>
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<p><b>Figure 13.</b> Blots of Prussian blue ferritin on nitrocellulose (20 uL samples) that are reacted with TMB (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.</p>
<p><b>Figure 13.</b> Blots of Prussian blue ferritin on nitrocellulose (20 uL samples) that are reacted with TMB (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.</p>
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<img src="https://static.igem.org/mediawiki/2013/8/89/UCalgary2013TRRecombinantPrussianBlueFerritin.png" alt="Creating Prussian Blue Ferritin out of our Own Ferritin" width="800" height="511">
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<p><b>Figure 14.</b> Measurements of the coloured substrate TMB (10 mg/mL) at 650 nm over a 600 second time period for our own Prussian blue ferritin and unmodified ferritin. Sample volume was 242 uL. Controls for this experiment include bovine serum albumin (1 mg/mL)and the substrate solution by itself.  Due to limitations on the protein available only one replicate was performed.</p>
<p><b>Figure 14.</b> Measurements of the coloured substrate TMB (10 mg/mL) at 650 nm over a 600 second time period for our own Prussian blue ferritin and unmodified ferritin. Sample volume was 242 uL. Controls for this experiment include bovine serum albumin (1 mg/mL)and the substrate solution by itself.  Due to limitations on the protein available only one replicate was performed.</p>

Revision as of 07:27, 27 September 2013

Prussian Blue Ferritin

What is Ferritin?

Ferritin is a ubiquitous iron storage protein found in both prokaryotes and eukaryotes allowing cells to keep iron in a soluble and non-toxic form. Ferritin across different species has very similar architecture and function. This is in despite of variations at the primary structure level (Harrison and Arosio, 1996). The 450 kDa protein shell consists of 24 subunits that can be composed of both heavy and light chains (Lawson et al., 1991) (Figure 1). Inside of this shell is room for an iron core composed of up to 4500 Fe (III) atoms stored as ferrihydrite phosphate (Ford et al., 1984). The goal of our project is to make use of this natural nanoparticle as both a scaffold and a reporter system.

Ferritin

Figure 1. Ribbon visualization of a fully assembled ferritin protein.

How can Ferritin be a Reporter?

Iron has naturally attracted attention for its ability to participate in Fenton chemistry. In this reaction iron acts as a catalyst in order to cause the disproportionation of hydrogen peroxide into oxygen-radical species (Figure 2). The resulting hydroxyl radical can cause the oxidation of common horseradish peroxidase substrates such as 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and 3,3’,5,5’-tetramethylbenzidine (TMB) to produce a colourimetric output. The standard iron core of ferritin however is not very suitable for catalyzing this process. The catalytic activity of ferritin can be increased by synthesizing ferritin with a magnetite core. The synthesis of this ”magnetoferritin” however is time consuming and is prone to disruption due to oxidation.

Fenton Chemistry

Figure 2. Chemical equation for the disproportionation of hydrogen peroxide into oxygen radical species using iron as a catalyst; Fenton chemistry.

An alternative to increasing the catalytic activity of ferritin would be to provide the necessary ferrous ions for the Fenton chemistry via the iron complex Prussian blue. This compound could act as a surface modification agent to give the ferritin core a Prussian blue surface. Prussian blue has previously displayed high catalytic activity and is believed to act as a source of negative charge at appropriate pH levels in order to yield an affinity with positively charged peroxidase substrates such as TMB (Zhang et al., 2010).

Synthesizing Prussian Blue Ferritin

The creation of Prussian blue ferritin is a relatively simple process that involves the surface modification of the iron core of normal ferritin. In order to accomplish this purified ferritin is combined with the compound potassium ferrocyanide under acidic conditions in order to produce the iron complex Prussian blue (Zhang et al., 2013). This chemical reaction can be seen below:

Prussian Blue Synthesis

Figure 3. Chemical equation for the synthesis of Prussian blue ferritin using potassium ferrocyanide.

The chemical process can easily be observed as there is a visible colour change from the colour of ferritin to the blue colour of Prussian blue ferritin (Figure 4). This reaction takes place overnight and then the Prussian blue ferritin is collected via dialysis. For this process we made use of commercial horse spleen ferritin. Based on our research we do not anticipate that this will display altered properties from that of the human ferritin that we will be producing in our E. coli.

Prussian Blue Synthesis

Figure 4. Comparison image of commercial ferritin to Prussian blue ferritin after the synthesis reaction. The synthesis reaction took place over a 12 hour time period.

Kinetic Testing of Prussian Blue Ferritin

The next step in moving forward with commercial Prussian blue ferritin as a potential reporter was to determine the kinetic characteristics of the catalyst. This ferritin is commercially purchased from Sigma-Aldrich. We chose this as a proof of concept for our characterization work as this ferritin is structurally very similar to the human ferritin we will be producing in our bacteria and does not have any different chemical capabilities. Our analysis is necessary in order to understand how our reporter will react over time in relation to different substrate concentrations. Based on previous data (Zhang et al., 2013) it had been seen that the catalytic activity can be fit to Michealis-Menten kinetic models allowing us an opportunity to evaluate the Prussian blue ferritin as a reporter. Our analysis composed of varying both the chromogenic substrate (TMB or ABTS) concentration and the hydrogen peroxide concentration.

Our kinetic analysis also included a comparison of Prussian blue horse spleen ferritin to regular horse spleen ferritin for both TMB and ABTS (Figures 5, 6). For both of the substrates we can see that normal ferritin has a very low catalytic activity. This is excellent news as it means that our Prussian blue modification is an effective modification to create a strong catalyst. It also means that the presence of unmodified ferritin is not active enough to pose a significant risk for producing spurious results in our system.

Prussian Blue Ferritin and TMB

Figure 5. Measurements of the absorbance of the 650nm light by the substrate TMB over a period of 600 seconds. 6 uL of 10 mg/mL substrate was used in a 242 uL reaction volume.Commercial Prussian blue ferritin ( 10 uL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 uL of 0.047 mg/mL sample). Negative controls are TMB and hydrogen peroxide, and TMB only. Standard error of the mean bars are based on a sample size where n=8. Substrate and hydrogen peroxide sample data is not clearly visible as it is in line with the substrate only data.

Prussian Blue Ferritin and ABTS

Figure 6. Measurements of the absorbance of the 415nm light by the substrate ABTS over a period of 600 seconds. 8 uL of 10 mg/mL substrate was used in a 242 uL reaction volume. Commercial Prussian blue ferritin ( 10 uL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 uL of 0.047 mg/mL sample). Negative controls are ABTS and hydrogen peroxide, and ABTS only. Standard error of the mean bars are based on a sample size where n=8.

Optimization of Prussian Blue Ferritin Reaction Conditions

The next step of our process was to determine the optimal conditions in which our commercial Prussian blue ferritin reporter could operate. This is a very important step in determining how we will move forward with creating a portable sensor device. The first variable tested was pH. For this variable we can see that the lower the pH the higher the activity of Prussian blue ferritin in the case of the ABTS substrate. As we raise the pH for this substrate we see that the activity drastically drops off. We did not test below a pH of 2 as the ferritin subunits would dissociate at this point. For the TMB substrate however we see that the activity has a much higher relative activity at a neutral pH compared to the ABTS substrate. This would be beneficial in our system as it means the substrate solution can be a lot friendlier to the other proteins present on the strip.

ABTS pH Optimization

Figure 7. pH optimization of commercial Prussian blue ferritin with ABTS. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 415 nm to detect the colourimetric change in a 242 uL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.

TMB pH Optimization

Figure 8. pH optimization of commercial Prussian blue ferritin with TMB. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 650 nm to detect the colourimetric change in a 242 uL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.

The next variable we tested was the effect of temperature on the commercial Prussian blue ferritin. For both substrates we can see that the as the temperature increases. Armed with this information we know that if can increase the incubation temperature of our system when we add the substrate solution we can potentially decrease the amount of time it will take our system to produce a signal from the presence of our target DNA.

ABTS Temperature Optimization

Figure 9. Temperature optimization of commercial Prussian blue ferritin with ABTS. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 415 nm to detect the colourimetric change in a 242 uL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.

TMB Temperature Optimization

Figure 10. Temperature optimization of commerical Prussian blue ferritin with TMB. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 650 nm to detect the colourimetric change in a 242 uL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.

Does Prussian Blue Ferritin Work on a Strip?

After doing the kinetic analysis of the Prussian blue ferritin it was necessary to think in terms of our actual device. There are many factors that we must balance in order to design the most optimal system. How much Prussian blue ferritin will give us a positive result that we can see with the naked eye? Which substrate will give us a result the fastest? What substrate colour is the easiest to see (Figure 11)?

Substrate Colours

Figure 11. Image of the colours of ABTS and TMB (10 mg/mL for both) after reacting with Prussian blue ferritin.

In order to test the Prussian blue ferritin in a strip environment we blotted varying concentrations of Prussian blue ferritin onto nitrocellulose; the material which will make up our strip system. From the results below it became readily obvious that TMB produced a much more distinct and identifiable colour compared to ABTS (Figures 12, 13). It was also able to detect the reaction of TMB when only 5 nanograms of Prussian blue ferritin was present. We anticipate that this detection limit could be improved given more refined methods of applying the protein to the nitrocellulose. We also decided based on the results of this experiment and previous experiments we have decided that TMB is the optimal substrate for our system. We could potentially explore more sensitive substrates to see if they interact with Prussian blue ferritin differently or are more sensitive with our system. We also tested Prussian blue ferritin in a basic prototype housing. You can see the results of this experiment on the Prototype page.

Prussian Blue Ferritin and ABTS on Nitrocellulose

Figure 12. Blots of Prussian blue ferritin on nitrocellulose (20 uL samples) that are reacted with ABTS (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.

Prussian Blue Ferritin and TMB on Nitrocellulose

Figure 13. Blots of Prussian blue ferritin on nitrocellulose (20 uL samples) that are reacted with TMB (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.

So can our Recombinant Ferritin become Prussian Blue Ferritin?

After our proof of concept efforts and once our own constructs of ferritin were purified it was necessary to see if this same treatment could be applied to bacterially produced human ferritin. The same synthesis reaction for to cause the Prussian blue surface modification of this ferritin was performed except that it was scaled down to match the concentrations of our ferritin. Our own Prussian blue ferritin was then exposed to the TMB substrate (Figure 14.). From the results we can see that the ferritin with the E-coil attached had excellent catalytic activity. The same ferritin with TALEs attached did not display as high of activity. While this result is disappointing it may be remedied with further optimization of the Prussian blue synthesis reaction. It also does not signify a major problem as we have multiple variants of our strip system and ferritin fused directly to TALEs is not a part that is absolutely necessary.

Creating Prussian Blue Ferritin out of our Own Ferritin

Figure 14. Measurements of the coloured substrate TMB (10 mg/mL) at 650 nm over a 600 second time period for our own Prussian blue ferritin and unmodified ferritin. Sample volume was 242 uL. Controls for this experiment include bovine serum albumin (1 mg/mL)and the substrate solution by itself. Due to limitations on the protein available only one replicate was performed.