| IIT-Roorkee - iGEM 2023

Sample Preparation

1)Sample preparation is the most crucial step in ITC. The initial quantities and masses of aptamer and lactoferrin are noted.

2)20mM Sodium Phosphate buffer(pH 7.4) is prepared from a stock solution of 55mM Sodium Phosphate and used as standard ITC buffer after taking in advice of P.hD in structural biology, Kunal Dhankar[1]. This was done as both DNA aptamers and protein are relatively large and act as macromolecules and thus remain stable in the phosphate buffer.

3)Stock solution of lactoferrin was produced. Molecular mass of human lactoferrin protein is 80kDa. 4.9 mg of human lactoferrin was weighed using a microbalance. Total moles of lactoferrin 61.25nMol. This was added in 200μL of phosphate buffer to produce a solution of 306.25 μM, but lactoferrin did not completely dissolve. This was further diluted by a factor of 5 to completely dissolve lactoferrin in solution. A stock solution of lactoferrin of 61.25 μM was obtained.

4)tock solution of DNA aptamer was produced using 3.19 mg of the aptamer ordered from IDT. The molar mass of aptamer was 180083.2mg/nmol. Aptamer was mixed with 100μL Nuclease free water and put in a water bath at 95 ℃ for 5 minutes for complete mixing. Concentration of stock solution was obtained at 1760μM. This was further diluted by a factor of 10 to create a working DNA aptamer working solution of 176μM.

Washing calorimeter

1)The Malvern Microcal PEAQ-ITC machine was washed first using Deionised water 3 times to ensure no contamination in experiment.

2)This was followed by washing using the phosphate buffer to allow proper loading of macromolecules.

3)All stock solutions were maintained at a pH 7.4 to maintain stability.

Protocol 1

1)40μL of DNA aptamer stock solution (176 μM) was loaded in the primary cell of ITC. Stock solution of Lactoferrin was diluted by a factor of 3.0625 to obtain a working solution of 20 μM. 3OO μL of the Lactoferrin working solution was loaded in a specialized ITC syringe and loaded into the sample cell of ITC

2)Reaction Parameters were set as follow

Protocol 2

Determination of Binding Affinities: ITC is widely used to measure the binding affinities between various biomolecules, such as proteins, nucleic acids, and small molecules. It can help identify the strength of molecular interactions, which is crucial in drug discovery and understanding biological processes

1)40μL of DNA aptamer stock solution (176 μM) was loaded in the primary cell of ITC. Stock solution of Lactoferrin was diluted by a factor of 1.53 to obtain a working solution of 40 μM. 3OO μL of the Lactoferrin working solution was loaded in a specialized ITC syringe and loaded into the sample cell of ITC

2)Reaction Parameters were set as follow

Data analysis

Data analysis was done using the MicroCal PEAQ-ITC analysis software.

References:

1) https://pubmed.ncbi.nlm.nih.gov/30929239/

Aptamer Reduction

1μmole thiolated aptamer was ordered as a single-stranded custom DNA oligo from IDT. Purified by Standard Desalting, the DNA arrived dry, 10.62mg (MW=18398.1) . Thiolated aptamers (Mod. Code: /5ThioMC6-D/) are shipped in their oxidized form, to protect the sulfur groups. The disulfide bond requires chemical reduction with Dithiothreitol (DTT) so the free thiol groups can bind to the AuSPE surface.

PROCEDURE

Lactoferrin (lF) Solutions:

10mg of lF was weighed and dissolved in 1ml PBS to form 10mg/ml primary stock soln, and then diluted to 20μg/ml, 10ml, secondary stock.

From this, 500μL dilutions were calculated and prepared in PBS for concentrations: 16μg/ml, 14μg/ml, 12μg/ml, 10μg/ml, 8μg/ml, 6μg/ml, 4μg/ml, 2μg/ml, 1μg/ml, 0.5μg/ml, and 0.1μg/ml.

Mercaptohexanol:

1.468μL of MCH was dissolved in 998.632μL of PBS to form 10mM, 1ml primary stock.

This was diluted to form 1mM, 10ml soln

Aptamer Soln.:

The 100 μM reduced aptamer solution was dissolved in PBS to form 5μM aptamer solution

Solution Preparation

Lactoferrin was purchased from Sigma Aldrich (L4040) and arrived as a solid powder. Biomarker (Lactoferrin) dilutions within and exceeding the required detection range (4.78-10.24μg/mL), Aptamer Soln., and Mercaptohexanol (which arrived as a pure liquid, MW=134.24 g/mol, density= 0.981 g/ml) for backfilling/blocking the electrode were prepared.

PROCEDURE

Lactoferrin (lF) Solutions:

10mg of lF was weighed and dissolved in 1ml PBS to form 10mg/ml primary stock soln, and then diluted to 20μg/ml, 10ml, secondary stock.

From this, 500μL dilutions were calculated and prepared in PBS for concentrations:

16μg/ml, 14μg/ml, 12μg/ml, 10μg/ml, 8μg/ml, 6μg/ml, 4μg/ml, 2μg/ml, 1μg/ml, 0.5μg/ml, and 0.1μg/ml

Mercaptohexanol:

1.468μL of MCH was dissolved in 998.632μL of PBS to form 10mM, 1ml primary stock.

This was diluted to form 1mM, 10ml soln.

Aptamer Soln.:

The 100 μM reduced aptamer solution was dissolved in PBS to form 5μM aptamer solution

Aptasensor Fabrication#1

AuSPEs are first cleaned and pre-treated to ensure uniform surfaces. The Self–Assembly-Monolayer (SAM) on an aptasensor consists of Aptamer + Backfilled MCH.

STEPS

Electrochemical Cleaning of Au-SPEs:
3 Cyclic Voltammetry Scans were performed in 0.5M H2SO4 (6, 10, and 6 scans in each run).
Scan Rate: 0.1 V/s, Range: -0.2V-1.2V.
It was rinsed with water and dried each time.
5μL of 5μM aptamer solution was drop-casted on the Working Electrode region of the SPE.
The electrodes were incubated for 16hrs.
The electrodes were washed in PBS to remove any excess unbound aptamers and then blocked with 1mM MCH.
They were incubated for another hour and then washed with PBS again (to remove excess MCH)

Aptasensor Fabrication#2

AuSPEs are first cleaned and pre-treated to ensure uniform surfaces. The Self–Assembly-Monolayer (SAM) on an aptasensor consists of Aptamer + Dithiothreitol + Backfilled MCH. Here, DTT + Aptamer were co-immobilized in a 1:10 ratio, while MCH was used as a blocking agent.

STEPS

Electrochemical Cleaning of Au-SPEs:
3 Cyclic Voltammetry Scans were performed in 0.5M H2SO4 (6, 10, and 6 scans in each run).
Scan Rate: 0.1 V/s, Range: -0.2V-1.2V.
It was rinsed with water and dried each time.
The immobilization solution consisted of 5μM, 250μL DNA + 500μM, 25μL DTT, effectively a mole ratio of 1:10.
5μL of the solution was drop-casted on the Working Electrode region of the SPE.
The electrodes were incubated for 16hrs.
The electrodes were washed in PBS to remove any excess unbound aptamers and then blocked with 1mM MCH.
They were incubated for another hour and then washed with PBS again (to remove excess MCH)

Preliminary Studies

Scan Rate Studies and Electrode Studies were conducted to verify the reproducibility and stability of the fabricated aptasensors. These are also done to select the stable electrodes for further Analyte Sensing/Electrochemical Response Studies.

STEPS

Scan Rate Study:

The SPE was connected to the Metrohm AUTOLAB PGSTAT101 Potentiostat which was interfaced with Nova 2.1.6 Software.
50μL PBS 7.4 with 5 mM [Fe(CN)6]3−/4− electrolyte was dropped on the electrode, such that the working region and counter electrode were completely covered.
Cyclic Voltammetry specifications were set to: -0.8V - 0.8V, 2 scans.
The scan rate was varied from 0.01 to 0.1 V/s at 0.01 increments and run, on the same electrode after its stability was established.
The second scan’s plot was recorded (as the first scan is for equilibration only) using Windower, and excel files were exported with the relevant E vs. I data.

Electrode Study:

A bare SPE was taken as a control and CV and DPV were run on both of them.
\ 50μL PBS 7.4 with 5 mM [Fe(CN)6]3−/4− electrolyte was used.
Cyclic Voltammetry specifications were set to: -0.8V - 0.8V, 2 scans, 0.05V/s scan rate.
DPV was set to the same potential window (-0.8V - 0.8V).
Relevant data was exported and plotted.

Electrochemical Response Studies

Analytes were sensed at prepared concentrations to understand and model the behavior of the Fabricated Biosensor.

STEPS

The SPE was connected to the Metrohm AUTOLAB PGSTAT101 Potentiostat which
was interfaced with Nova 2.1.6 Software.
50μL PBS 7.4 with 5 mM [Fe(CN)6]3−/4− electrolyte was dropped on the electrode,
such that the working region and counter electrode were completely covered.
Each sample concentration of lactoferrin was dropped onto their respective electrode
and the electrochemical processes were performed almost immediately after each.
Cyclic Voltammetry specifications were set to: -0.8V - 0.8V, 2 scans, 0.05V/s scan rate .
DPV was set to the same potential window (-0.8V - 0.8V) and DPV plots were used for sensing studies.
Relevant data was exported and plotted.