Team:Patras/Implementation

Proposed Implementation

According to the latest World Health Organization’s (WHO) study, the first cause of death worldwide is cardiovascular diseases. In particular, one out of three deaths in the developing countries is due to them, while in developed countries may reach 80%. Remarkably, one out of ten people aged between 30 and 70 will die from such a disease (including heart attacks and strokes) due to the lack of balance in the blood’s LDL cholesterol levels.

The category of drugs administered to regulate LDL levels is called statins. However, 1 out of 3 patients does not appear to have the expected results because of several side effects such as myopathy and liver associated problems. So, our diagnostic tool could be an effective way to overcome the issues mentioned above. The BentoLab – AI system allows doctors to provide excellent dosage adjustment services to their patients suffering from cardiovascular diseases, such as hypercholesterolemia.

Physicians, like cardiologists, with a lack of laboratory knowledge, can perform the test quickly and effectively. Without previous laboratory experience, three medical students testified our BentoLab-AI system to ascertain its accuracy and convenience. George Vasilagkos, Kanellos Spiliopoulos, and Athanasios Vagionis checked out a trial version of it just following the step-by-step guidance provided to them by our system. At this point, we should underline that each student performed the test on a different day. Thus we had enough time to use their feedback and enhance the instructions. Our goal was to improve the step-by-step guidance and make BentoLab – AI system understandable for every user.

Step-by-step Guidance

Start by clicking the arrows below!

DNA purification with NucleoSpinⓇ Blood
(For more details you can use the booklet provided from the DNA kit extraction)

1. Before starting the preparation for the DNA purification from whole blood:

• Wear double gloves.
• On the first use of the extraction kit, prepare Buffer B5, by adding 48 mL ethanol.
• On the first use of the extraction kit, prepare Proteinase K, by adding the indicating volume of Proteinase K Buffer PB.
• Set the Thermoblock to 70 ℃
• Use an 1.5 mL Eppendorf, and with a pipette add (number of blood samples * 50) μL Elution Buffer BE.
• Place the Eppendorf with the BE in the Thermoblock to be preheated.
• Prepare the work surface, by laying absorbent paper, prepare a container with bleach where all the tips and Eppendorfs that will be used during the isolation of the DNA will be discarded.
• Sanitize the pipettes with ethanol.
• All the pipette tips are sterilized and is essential not to make contact with anything else before use.
• Fill a container with water and place the Blood samples to thaw.
• Use as many Eppendorfs as the number of examined blood samples.
• On each Eppendorf’s cap write the number of each blood sample.

2. Use a pipette of 1000 μL (blue colour), filter tips and add on each Eppendorf 200μL blood of each blood sample, respectively.

* Advice: Before adding the blood into the Eppendorf mix “up and down”. Use a different filter tip, while adding each blood sample on its Eppendorf.

3. Use a pipette of 200μL (yellow colour), non-filter tips and add 25μL Proteinase K on each Eppendorf.

* Advice: Mix “up and down” the Proteinase K, every time you add it on each Eppendorf. Use a different tip in every add. We add the Proteinase K into the main volume of the blood.

4. Use a pipette of 1000 μL (blue colour), non-filter tips and add on each Eppendorf 200μL Buffer B3.

* Advice: You can use the same tip if it does not come in touch with another surface.

5. Vortex each Eppendorf vigorously (10-20 s) on the vortex device.

6. Place the Eppendorfs on Thermoblock to be incubated at 70 ℃ for 15 min.

7. Use a pipette of 1000 μL (blue colour), non-filter tips and add 210μL ethanol (96-100%) to each sample.

* Advice: You can use the same non-filter tip to add the Ethanol if it does not come in touch with another surface.

8. Vortex each Eppendorf vigorously (10-20 s) on the vortex device.

9. For each Eppendorf, take one NucleoSpinⓇ Blood Column placed in a Collection Tube and load the sample. Write on the cap of the Blood Column the code of the sample.

* Advice: Use a pipette of 1000 μL (blue colour),filter tips and regulate the volume on 600 μL to be sure that all the volume of the sample will be loaded. Use a different filter tip to load each sample.

10. Centrifuge 1 min at 8x kg by placing them anti-diametral on the centrifuge in BentoLab.

* Advice: If the samples are not drawn through the matrix completely, repeat the centrifugation at higher g-Force (<15.000 x g ).

11. Place the NucleoSpinⓇ Blood Column into a new Collection Tube (2mL) and discard the previous Collection Tube with flow-through on the bleach container.

12. Use a pipette of 1000 μL (blue colour), non-filter tips and add 500μL Buffer BW.

* Advice: You can use the same tip to add the BW if it does not come in touch with another surface.

13. Centrifuge 1 min at 8x kg, by placing them anti-diametral on the centrifuge in BentoLab.

14. Place the NucleoSpinⓇ Blood Column into a new Collection Tube (2mL) and discard the previous Collection Tube with flow-through on the bleach container.

15. Use a pipette of 1000 μL (blue colour), non-filter tips and add 600μL Buffer B5 on each sample.

* Advice: You can use the same tip to add the B5 if it does not come in touch with another surface.

16. Centrifuge 1 min at 8x kg, by placing them anti-diametral on the centrifuge in BentoLab.

17. Place the NucleoSpinⓇ Blood Column back into the Collection Tube and discard the previous Collection Tube with flow-through on the bleach container.

18. Centrifuge 1 min at 8x kg, by placing them anti-diametral on the centrifuge device in BentoLab.

19. Place the NucleoSpinⓇ Blood Column back in an Eppendorf and discard the Collection Tube with flow-through on the bleach container.

20. Use a pipette of 1000 μL (blue colour), non-filter tips and add 50μL preheated Buffer BE (70oC).

* Advice: You can use the same tip to add the BE if it does not come in touch with another surface.

21. Let the Eppendorfs at room temperature for 1 min.

22. Centrifuge 1 min at 8x kg, by placing them anti-diametral on the centrifuge in BentoLab.

23. Each Eppendorf contains the DNA from each blood sample.

24. Nanodrop. According to our experiments, all of the 81 DNA samples that extracted with the NucleoSpinⓇ Blood (Macherey - Nagel) Kit, when measured in nanodrop, resulted in concentrations with a range from 50 to 100 ng/uL. Therefore, any other sample extracted with the same method, its concentration is expected to be in this range (50 -100 ng/uL) and there is no need to be diluted.

25. PCR
Before the PCR

• Wear news gloves
• Prepare the work surface, by cleaning it with ethanol and laying absorbent paper
• Prepare the pipettes of 200 μl, 20 μl and 10 μl
• Sanitize the pipettes with ethanol.
• Prepare two Eppendorfs and write on the one cap Master Mix W and on the other Master Mix M.
• Prepare the PCR tubes: Use as many as the DNA samples + 1 more for the Blank sample.
• Write on each tube’s cap the name/ number of each DNA sample followed by the w. (These will be the tubes where the Master Mix W will be loaded).
• Prepare the PCR tubes: Use as many as the DNA samples + 1 more for the Blank sample.
• Write on each tube’s cap the name/ number of each DNA sample following by the m. (These will be the tubes where the Master Mix M will be loaded).
• Place the PCR tubes on the red base and leave them alongside.
• Thaw the reagents by rubbing them between the palms, except from the Taq Polymerase (Important: Remove the Taq from the ice at the time of addition).
• As soon as they are defrosted, centrifuge them at 2x kg and stop the time after 10 sec.
• Take the orange icebox and place the reagents.
• Place the Eppendorfs of Master Mixes on the orange icebox and prepare them there.
• Calculate the appropriate concentration of PCR reagents by multiplying the given concentrations for one sample, on the following table with A (A= the number of samples + 1 blank sample + 1 extra)

PCR reagents	Concentration for one (1) sample	Concentration for N samples
Distilled H2O (ddH2O)	17 μl	17 * A μl
Buffer Solution	2.5 μl	2.5 * A μl
MgCl2	2.5 μl	2.5 * A μl
Forward primer wild type (Fw) / Forward primer mutant (Fm)	0.75 μl	0.75 * A μl
Reverse primer	0.75 μl	0.75 * A μl
dNTPs	0.25 μl	0.25 * A μl
Taq Polymerase	0.25 μl	0.25* A μl

26. Prepare the Master Mix W: Add the reagents with the according order (from higher to lower volume): ddH2O, Buffer Solution, MgCl2, Forward primer for the Wild type (Fw), Reverse primer, dNTPs, Taq Polymerase.

* Advice: Use non-filter tips. Remove the Taq Polymerase from the ice at the time of addition. When adding each reagent, use a different non-filter tip and mix "up and down" before taking the quantity with the pipette.

* Important: Pour the quantity without depressing the second rung of the pipette!!!

27. Prepare the Master Mix M: Add the reagents with the according order (from higher to the volume): ddH2O, Buffer Solution, MgCl2, Forward primer for the Mutant (Fm), Reverse primer, dNTPs, Taq Polymerase.

* Advice: Use non-filter tips. Remove the Taq Polymerase from the ice at the time of addition. When adding each reagent, use a different non-filter tip and mix "up and down" before taking the quantity with the pipette.

* Important: Pour the quantity without depressing the second rung of the pipette!!!

28. Centrifuge the master mixes at 2x kg and stop the time after 10 sec., by placing them anti-diametral on the centrifuge in BentoLab.

29. Take the purple icebox and place the PCR tubes.

30. On the PCR tubes w divide the Master Mix W, by adding in each tube 24 μL.

* Advice: Use a pipette of 200 μL and non-filter tips. Mix “up and down” every time you share. You can use the same tip if it does not come in touch with another surface.

31. On the PCR tubes m divide the Master Mix M, by adding in each tube 24 μL.

* Advice: Use a pipette of 200 μL and non-filter tips. Mix “up and down” every time you share. You can use the same tip if it does not come in touch with another surface.

32. Thaw the DNA samples. Centrifuge them for 10 sec and place them on the orange icebox.

33. Use a pipette of 10 μL, filter tips and add 1 μL of each DNA sample on the according PCR tube.

* Advice: Use a different filter tip every time you add the DNA. Mix “up and down” the DNA before adding on the PCR tubes.

34. Use a pipette of 10 μL, non-filter tips and add 1 μL of ddH2O on the blank PCR tubes.

* Advice: You can use the same non-filter tip if it does not come in touch with another surface.

35. Centrifuge the PCR tubes, by placing them anti-diametral on the centrifuge device on BentoLab

36. Place the PCR tubes into the PCR device in BentoLab and adjust the proper PCR conditions and PCR cycles at 30 cycles. (Estimated time: 152 min)

Temperature	Time
95°C	5 min
95°C	30 sec
65°C	50 sec
72°C	120 sec
72°C	10 min

37. Electrophoresis
While waiting the PCR to finish, start preparing the agarose gel so to be ready as soon as the PCR ends.

38. Weigh 0.5 gr of agarose and put it in a flask.

Electrophoresis

39. Add 50 mL of Buffer TBE 1x in flask.

40. Place it at high temperature in a microwave oven to be dissolved, until the solution becomes clear.

* Advice: Due to the high temperature, use a double wrapped absorbent paper and place it around the flask’s mouth.

41. Periodically, remove the flask from the microwave oven and shake it.

42. When agarose has been dissolved, add rapidly of gel stain and shake it.

43. Adjust the gel on the electrophoresis device on the BentoLab.

44. Place the electrophoresis combs to create wells and leave it for at least 30 min to solidify.

45. Once the gel is ready, remove the rubber barriers and the electrophoresis combs and add Buffer TBE 1x to cover the entire surface.

46. Add 5 μl of 100 bp ladder on the first well.

47. Place each PCR product on a different well on the gel placed on the Electrophoresis device.

* Advice: Place the PCR products on the wells with the following system: Sample A w, Sample A m, Sample B w, Sample B m, etc , Blank Sample w, Blank Sample m.

48. Run the electrophoresis.

49. After the electrophoresis ends, in dark room, place the specially designed box on the BentoLab and turn on the Blue light to see the results.

50. If the ladder has not been opened, as expected, run the electrophoresis few minutes extra.

51. Observe again the results on the Blue light, with the same way as described on step 50.

52. If the results are satisfactory, place your phone just above the opening of the specially designed box.

53. Upload the photo on the platform.

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Today, clinic doctors are unaware of genetic tests’ availability relating to treatment decisions and how to interpret them. The lack of familiarity with genetic tests may be one reason for their rare integration into clinical practice. Thus, students associated with health sciences need to be informed on the Pharmacogenomic field’s usefulness as part of their school’s curriculum.

We also propose to microbiologists the use of our diagnostic system to enrich their diagnostic techniques. Being experts in such techniques, it would be effortless for them to use the system even without the provided step-by-step assistance.

The Ministry of Health and other relevant governmental agencies can make the BentoLab – AI software system available to public hospitals not only in metropolitan areas but also in the remote ones, taking advantage of its portability. Through our system, the time needed for genomic analysis is decreased from 5-10 days to only 5-6 hours, while the cost is extremely reduced compared to existing methods.

The high accuracy that the BentoLab-AI system provides makes it a great option even for Pharmaceutical Company’s R&D departments. By using it, they can personalize the dosages of drugs that they produced and simultaneously avoid the supernumerary production of drugs.

We envision BentoLab as a molecular diagnostic device, supported by an Artificial Intelligence software that would show to physicians what dosage of every drug – and not only statins - a patient should take. So, we will achieve personalized medicine, cheaper, faster, and more accurately than any other method tried before.

According to the World Health Organization, 33% of deaths in the developed countries come due to cardiovascular disease, while this percentage may reach 80% in countries with medium and high incomes, such as North America and Europe. Specifically, Europe has the highest rate of people with raised cholesterol (54% of both sexes), followed by America with a percentage of 48% for both sexes. Especially in Greece, 46% of men and 40% of women have general serum cholesterol levels >200mg/dl. Of them, 40% of men and 30% of women were ignorant of their condition. Of those, 31% of men and 20% of women receive pharmaceutical treatment, mainly statins. This markets’ growth can be majorly related to the unhealthy lifestyle adopted by the citizens in these regions and the inductive increase of the obesity rates. In such areas, our project would be extremely beneficial for patients taking statins as it can be used for the elimination of their side effects.

Although our project seems harmless, there are some safety aspects that we should consider. It has to be clarified that the following aspects are not related to laboratory equipment, manipulations, and techniques, which are published in Safety forms.

We specifically concentrated on bioethics, which is the study of typically controversial ethics brought about by advances in biology and medicine. It is also moral discernment as it relates to medical policy, practice, and research. The term Bioethics (Greek bios, life; ethos, behavior) was coined in 1926 by Fritz Jahr. However, the fundamental principles announced in the Belmont Report (1979)—namely, respect for persons, beneficence, and justice—have influenced the thinking of bioethicists across a wide range of issues.

Our team decided to analyze the bioethical implications of our project to prove that not only does it not pose any risk for our society, but it can also prove to be beneficial. The field of synthetic biology could be both a useful and harmful tool - like a knife - depending on the utilization that someone may do and someone’s ethos.

We believe that Ethics is an integral part of the fields of Science and Technology. There are many different factors to be considered when trying to approach the ethical aspects of them, especially when we attempt to do that for the newly emerged field of Synthetic Biology. While searching the scientific literature, we realized various definitions of Synthetic Biology, but not entirely adequate. One that could partially be satisfactory is that Synthetic Biology is more than just another field in Biology or Engineering. It’s a fusion of these two, in ways that can help you design and build entirely new systems or copy the already existing ones artificially.

Our priority was to ensure patients’ consent for blood sampling. This was important to be assured as we needed the blood not only to conduct our experiments but also to train the algorithm. This procedure is done through a written document, and it requires specific information such as in which place the samples will be stored, who will have access to our data, and how they could be used. This consent should be legal and follow a specific procedure.

Bellow you can find the Consent Form legally accepted in Greece.

Consent Form - Click here to view our Consent Form!

Finding a specialist lawyer to guide us through our future steps wasn’t as easy as it sounds! Vasileios Karkatzounis, a legal engineer specialized in the proper use of Artificial Intelligence, highlighted all the legal obstacles we may face while trying to turn our project into a sustainable business plan. Initially, he informed us about the regulatory system and guidelines published in 2018 by the European Council whilst he urged us to read them through, especially those referred to as personal data, used for the AI system. He underlined the necessity to create a confirmation form in order to be within the law while using the health data to develop the BentoLab-AI system. The reason and aim of using these data, the duration, the results, the owner, and the legal base system should be included in this form. In addition to these, all volunteers should be elaborately informed about the procedure that will be followed and how their personal data and the results will be used. Of course, it should be clarified from the beginning to all the participants that our project’s supervisor is the University of Patras. Last but not least, his most purposive advice was to clarify that the AI system will be used for physicians’ assistance, and it won’t replace their decision.

The general public’s scaremongering is also a crucial issue that should be mentioned. There is a widespread view that AI is an ambiguous subject because of the retention of personal data. So, we tried to solve this problem through the document we provided to each volunteer. In addition, no GDPR issue is arising since our system needs only a photo of the electrophoresis results and no other data. The people should realize that Synthetic Biology, Pharmacogenomics, and AI can help solve societal issues using logical arguments. We should not be afraid of contrasting opinions since they can result in fruitful discussions.

Moreover, a significant problem that we should check is the environmental risks that our project may pose. Dr. Patrinos highlighted the fact that there is a slightly increased risk of non-predictable and undesired interactions of our products with the environment, such as wasted plastic dips or wasted pigments. To minimize this danger, he suggested that we are careful with our lab waste. Recycling all the used plastic dips and tubes and dismissing the wasted organic and inorganic solutions in the predefined containers for this job to be safely released to the environment by the experts would be the right steps towards this direction.