| IIT-Roorkee - iGEM 2023

Protocol 1:

In the first run we started with initial concentrations of DNA aptamers at 176.41 uM and initial concentration of lactoferrin at 20mM. Larger amount of aptamer was taken to ensure sufficient number of DNA aptamer molecules were present to bind to the protein

Observation:

In the Differential Power vs Time graph, The peak very quickly shifts from exothermic region (below zero DP) to endothermic region(above zero DP), that is the reaction changes from exothermic to endothermic after just two injections (two peaks).

△H vs Molar ratio graph is initially unable to fit the starting two injections on the plot. After using the fit function in the Microcal PEAQ-ITC data analysis software, the plot is finally able to fit all points in a satisfactory manner

The reaction parameters obtained were as follow

ITC experiment produced the following graph

b) Inference:

Kd value of 1.45 x 10^-9, which signified strong bonding between the aptamer and protein. A total of 8 aptamer molecules were binding to a single lactoferrin molecule, indicating insufficient protein quantity.

Despite binding occurring, the reaction becomes endothermic very quickly indication that protein starts denaturing with only a small quantity of Aptamer

It was understood that protein quantity must be be increased to avoid denaturing of protein by aptamer quickly

Protocol 2

In the second run we started with initial concentrations of DNA aptamers at 176.41 uM and initial concentration of lactoferrin at 40mM. Larger amount of lactoferrin was taken to ensure sufficient protein molecules available for aptamer binding and avoid denaturing similar to Protocol

a)Observation:

In the Differential Power vs Time graph, The peak shifts from exothermic region (below zero DP) to endothermic region(above zero DP) after some injections.

After using the fit function in the Microcal PEAQ-ITC data analysis software, the reaction parameters obtained were as follow:

ITC experiment produced the following graph

Inference :

The Kd remained the same and binding still occurred,A total of 4.03 aptamer molecules were binding to a single lactoferrin molecule

△H vs Molar ratio graph fits all points and is balanced. Strong Binding is observed.

Results and Analysis

SCAN RATE STUDY

1. Slightly unstable peak shape on the right end above 60mV/s
2. Proceed with 50mV/s (also recommended in literature)

ELECTRODE STUDY

1. Cyclic Voltammetry current shows barely any significant changes.

2. Aptamer Immobilisation on the SPE shows significant rise in current in Differential Pulse Voltammetry, the more sensitive and selective process. DPV shows better signal to noise ratio too, as it can distinguish between background currents. We chose to go ahead with DPV for further studies.

3. As shown in subsequent sensing studies, the Aptamer/MCH-Immobilised SPE reproducibly showed an increased current between the 55 and 65μA range, successfully indicating aptasensor fabrication. The Aptamer/MCH/DTT/SPE showed even higher DPV values.

ELECTROCHEMICAL RESPONSE STUDY#1

1. The black curve labeled DPV corresponds to just the Aptamer/MCH/SPE surface = 55μA (stable aptasensor).

2. The corresponding DPV curves on trying to detect 10μL analyte in each electrode clearly reach saturation within the very first concentration (0.1μg/mL) itself.

3. All the available aptamers for binding on the electrode surface seem to have been bound to analyte (lactoferrin).

4. We decided to proceed with lower analyte volumes (3μL) for subsequent studies.

ELECTROCHEMICAL RESPONSE STUDY#2

The Aptamer/MCH/DTT/SPE Sensor shows a significantly higher current peak (72μA).

Saturation is reached much quicker despite using only 3μL of analyte.

An explanation for this is the ratio of DTT:Aptamer used was too high, leading to too few Aptamers binding to the electrode and more DTT binding in the available surface area on the electrode. Therefore there being less Aptamer for the Lactoferrin to bind to.

The minute difference between saturation current and aptasensor current indicates that it isn’t possible to conduct any meaningful sensing with the aptasensor within that range.

ELECTROCHEMICAL RESPONSE STUDY#3

The top most green curve is the Aptamer/MCH/SPE Sensor surface with no analyte= 55μA (stable electrode)

Eventually, using the Aptamer/MCH/SPE Sensor and 3μL analyte for all concentrations, a clearly and significantly decreasing trend was observed from 0.1-16μg/mL.

By 14μg/mL, clearly saturation current (around 40μA) was beginning to be reached again, as the subsequent differences between currents are growing smaller, as indicated in the following zoomed graph:

It is inferred that the conformational changes in the aptamer on binding to Lactoferrin blocks the active sites on the sensor surface or modifies its electrochemical properties such that current steadily decreases with more binding.

LINEARITY STUDY

On plotting our obtained current values against their corresponding concentrations, we were able to establish a linear relationship between the concentrations 4μg/mL and 14μg/mL with an R-Square Value of 0.98801 which is considered excellent and establishes a reliable linear correlation.

This range falls well within the required range discussed for Alzheimer’s patient’s saliva (4.78μg/mL of Lactoferrin) versus a healthy person’s saliva (10.24 μg/mL)

NOTE

We encountered issues with the stability of several electrodes/fabricated sensors that were discarded due to rapidly changing DPV plots (perhaps due to leaching of the aptamers into the buffer).

The electrodes used were the ones that produced stable and constant DPV Values over repeated runs.

Work needs to be done for creating reproducible results with new batches of electrodes to ensure the stability of electrodes and a reliable immobilization protocol.

One possible explanation for the leaching was that the discarded electrodes were mostly from a second batch of immobilization that was done a day after the reduction of the aptamers. Even though they were stored at -20C it is possible that they resolved back into their oxidized form with the disulfide bond, hindering effective immobilization