Proof of Concept

Solve the problems step by step

Overview

Because we cannot use living porcine eyes to verify the function of our contact lenses, we used step-by-step experiments to verify that our contact lenses are effective and useful in the treatment of glaucoma in the future. The proof of concept is divided into three parts. In the first step, we used Digital Image Correlation (DIC) to verify that the change in IOP can cause the deformation of the contact lens, and calculated the concentration of the cavity change. With this concentration change, we used the enzyme kinetics model in the second step to calculate the amount of NOS produced by the engineered bacteria. Finally, because NOS will convert L-arginine into NO, we measured the NO concentration in the aqueous humor of the porcine eyes to know whether NO successfully diffused out of the cornea and effectively acts on the trabecular meshwork.

Step 1

IOP simulation experiment

To test the amount of contact lens deformation on the cornea, we designed an IOP simulation experiment with a porcine eye. Based on Pascal's principle of fluid mechanics, the pressure produced by fluid in an enclosed environment will be proportional to the height difference between the fluid surface and the target site. By changing the drip bag’s height, water pressure will directly increase IOP in the porcine eye, enabling precise control of IOP for experiments^[1]^[2].

We used the porcine eye from the abattoir. All the eyeballs should not be blanched by the hot water and should be stored at 0℃. First, we used pin to fix it on the styrofoam. Then we used the trocar to pierce into the limbus between the cornea and conjunctiva and infused normal saline into the aqueous humor for controlling the intraocular pressure. By doing so, we were able to start establishing the relationship between the IOP and the deformation of eyeballs, which is obtained from the digital image correlation system (DIC).

Fig. 1. The device of IOP simulation experiment.

The data analyzed by DIC are e_xx (the strain along the X axis), e_yy (strain in the Y axis), e_xy (the shear strain tensor-note that this is equal to half the engineering shear strain.)^[3]

Fig. 2. The mathematical expression of e_xx e_yy e_xy.

Note that u, v represents the raw displacement between the reference image and the given image in x- axis and y-axis, respectively.

And the data we get is linear. Fig. 3 indicates a graph of e_xx deformation - IOP. Likewise, e_yy and e_xy have similar experimental results.

Fig. 3. Shows that the relation of IOP increment with respect to strain is linear. (N=3)

According to the formula in Fig. 4, we can calculate the volume change, and use the volume calculated from the model to calculate the concentration change.

Here, we considered the general volume change ratio since the equipment we used to observe the deformation, namely DIC, disable us to obtain the thickness difference of contact lens.

Fig. 4. The mathematical expression of calculating the volume change ratio.

As shown in Fig. 4, the pressure change of 1mmHg will reduce the volume of the contact lens by 0.29% and cause a concentration of 0.29%.

Compared with the 1% deformation calculated by the model, the data results obtained by DIC are smaller. Given that the samples we adopted for the experiment are porcine eyeballs obtained from the abattoir, which the coagulative necrosis had occurred, leading to the denaturation of the cornea^[4]. It is imaginable for the results to be different from the calculation from model.

Step 2

IPTG induction experiment

To prove that different concentrations of IPTG can induce our bacteria to produce different amounts of NOS, we test the NOS amount with NOS assay kit.

For preparation, we cultured the bacteria containing a plasmid with an IPTG inducible promoter and NOS gene. After culturing overnight, we induced bacteria with different concentrations of IPTG for different periods of time. We then adjusted the OD₆₀₀ to one and disrupt the cell.

The substrate, mainly L-arginine and NADPH, was added to the disrupted cell. After twenty minutes of incubation, the L-arginine was transformed into NO. The buffer of NOS assay kit then transformed NO into Nitrite for Griess reagent to interact with.

We measure the OD₅₄₀ and convert it into NO concentration with a standard curve.

Fig. 6. NOS induced by IPTG with different concentrations at different induced times.

Step 3

NO difusion experiment

The third step of the proof of concept is to prove that nitric oxide can effectively pass through contact lens and cornea, and successfully enter aqueous humor.

After lysising the bacteria, a solution containing NOS is obtained and is dripped on the contact lens together with the L-arginine solution. Then the L-arginine will be decomposed while the nitric oxide being produced, which can penetrate into the aqueous humor. We collect the aqueous humor by the needle and convert it into nitrate for the measurement of NO kit, and the result will be compared with that of the control group. Fig. 7 showed that the [NO] in the aqueous humor of the experimental group is 6 times as much as that in the control group.

Fig. 7. The diffusion of nitric oxide from contact lens to aqueous humor.

For the porcine eyes of the experimental group, we put the contact lens on the porcine eyes, dripped the reaction solution (bacterial liquid of NOS + L-Arginine), put into a 37⁰C incubator, waited for 1.5 hours, extract the aqueous humor, and then measured it with NO kit. The control group was the same batch of porcine eyes as the experimental group, it was put into a 37⁰C incubator for 1.5 hours before extracting the aqueous humor, and then measured them with NO kit. As for the background value used for calibration, we placed the bacterial liquid NOS + L-Arginine in a 96-well plate, waited for 1.5 hours, and then measured with NO kit.

Fig. 8. Schematic diagram of aqueous humor extraction.

To simulate the real condition of wearing the contact lens on the eyes as much as possible, we use our self-made contact lens and drop the reaction solution in the chamber to cover a circular plastic film, which can prevent the diffusion of nitric oxide into the air. In the future, our contact lens will also use similar methods to improve our treatment effects.

Biofilm Confirmation

We want to ensure our bacteria can bind to contact lens, so we put a contact lens into a liquid culture. We preculture BL21(DE3) as control of BL21(DE3) with plasmid NOSDA 3ml overnight and adjust OD₆₀₀ to 0.2 for 5ml, and we put the contact lens into the culture incubate without shaking for two days. Fig. 9 showing that bacteria overexpressed csgD can adhere to the contact lens, while the control groups can not. So we know that our bacteria can attach to the contact lens well.

Fig. 9. (A)Contact lens in bacteria with plasmid NOSDA. (B)Contact lens in bacteria with control plasmid.
The arrow is pointed to the contact lens.

Eye Screen

To validate the function of Eye Screen, we adopted a gravity model to control the IOP of porcine eyeballs using the trocar system via microincision vitrectomy surgery. By adjusting the height of the saline drip bag connected to the eyeball, we can measure the IOP by calculating the difference in height of the saline and the eyeball. For more detail please link to Eye Screen.

References

Chen G-Z, Chan I-S, Leung LKK, Lam DCC. Soft wearable contact lens sensor for continuous intraocular pressure monitoring. Medical Engineering & Physics. 2014;36(9):1134-1139.
Zhang J, Zhang Y, Li Y, et al. Correlation of IOP with Corneal Acoustic Impedance in Porcine Eye Model. BioMed Research International. 2017;2017:1-6.
Strain Tensors and Criteria in Vic. Retrieved from http://correlatedsolutions.com/support/index.php?/Knowledgebase/Article/View/2/0/strain-tensors-in-vic
Nabeeha Khalid, Mahzad Azimpouran. Necrosis. Nih.gov. Published May 10, 2020. Accessed October 24, 2020.

Team:NCKU Tainan/Proof Of Concept