Proposed Implementation

Proposed Implementation

Abstract

POIROT, developed by UTokyo 2024, is capable of detecting and quantifying very small amounts of the glaucoma biomarker microRNA. The main characteristics of the reaction are as follows:

  • A series of reactions proceeds at isothermal temperature (37 ℃)
  • High specificity
  • The detection results are easily recognizable at a glance.

These features were designed with the POIROT user in mind first. Our goal is to create a device that everyone can use at home. Therefore, in addition to Human Practices to academia and social implementation experts, feedback was obtained many times through Education to actively incorporate the opinions of future POIROT users.

Initially, POIROT's detection target was the glaucoma biomarker miRNA, but the system's flexibility has made it possible to amplify and quantify a variety of miRNAs with only minor changes to the sequences involved in amplification. POIROT will bring further development to clinical testing through synthetic biology.

For Glaucoma

Background

Glaucoma is one of the leading causes of blindness in the world, and Japan as well. One in twenty people over the age of 40 and one in ten people over the age of 60 are said to have glaucoma in Japan 1. It is possible to detect glaucoma at an early stage by visiting an eye doctor on a regular basis. By starting appropriate treatment at an early stage, the progression of symptoms can be delayed and blindness can be prevented. However, many busy modern people cannot afford to go to an eye doctor for regular checkups 2.

End User Analysis

During the initial design phase of POIROT, we conducted Human Practices with ophthalmology clinicians, researchers, and glaucoma patients, and found that there was demand for early detection of glaucoma. The users of POIROT are the public and do not necessarily know much about glaucoma or have specialized knowledge or skills. This means that our first priority must be that everyone can use POIROT. Therefore, when designing the product, we must gather input not only from experts, but also from the public and integrate them into the project.

Currently, the diagnosis of glaucoma is made at most eye doctors through continuous measurement of intraocular pressure, fundus examination, and visual field testing. Many people stop visiting eye doctors due to the time and financial burden of the visit and the cost. Therefore, aiming to know more about the need for glaucoma examinations to be conducted in the home, we conducted a questionnaire survey targeting the public.

  • General Public's Perception of Glaucoma
    • We surveyed the general public's perception of glaucoma itself and the frequency of visits to eye doctors by conducting a questionnaire on the Internet and during Education, and received nearly 250 responses.
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    Figure 1.

    The survey results indicate that the name glaucoma is known, but not much is known about its treatment and diagnosis.
  • What the public would like to see in a glaucoma test device
    • When surveying the public's awareness of glaucoma, we also investigated what they expect for a future glaucoma test device. It helped us understand what elements are desired in a testing device.
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Figure 2.

What is most required is ease of use. As per our original philosophy, we reaffirmed that there is great significance in developing a test device that can be used by everyone.

These results indicate the need for a diagnostic aid for glaucoma that can be used at home. The survey revealed that most of the public has misconceptions about glaucoma or is not familiar with it in the first place. This was also pointed out to us when we conducted a HP with a glaucoma patient group, and we learned that some patients become depressed and overly nervous about their future because of the diagnosis of glaucoma.

We as scientists must take responsibility for what we create. If we merely develop a system for early glaucoma detection, we will not promote a proper understanding of glaucoma and will only leave the user with an emotional burden in life. We have a responsibility to promote a correct understanding of glaucoma itself, that with proper treatment, the progression of glaucoma symptoms can be suppressed.

In addition, in response to the need for user-friendliness, we focused on tear fluid, which can be collected minimally invasively and has a stable composition, as the biological sample required for the test. Since miRNAs are a good indicator of in vivo conditions, we decided to use the glaucoma-specific biomarker miRNAs in tear fluid for the test.

Selection of Biomarker

The specimen candidates were narrowed down with a view to making the glaucoma detection device easy to use at home. Common specimens include blood, oral mucosa, and urine. Other samples include hair, aqueous humor, and tear fluid.

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Figure 3. MiRNA Selection

The stability as a specimen, ease of collection, and invasiveness were taken into consideration. We also conducted Human Practices with eye doctors and specialists in tear ducts, tear fluid, and POCT, and concluded that tear fluid is an appropriate specimen for this study.

It has been reported that glaucoma damages the optic nerve, which in turn destroys optic nerve cells and releases miRNAs, and thus the tear fluid contains the glaucoma biomarker miRNA (Sabirzhanov et al., 2014) 3. Although the location of the optic nerve is separated from the location of the tear fluid by the biological membrane and the released miRNAs do not flow directly into the tear fluid, we still turned our attention to the tear fluid. However, there are not many reports on miRNAs in the tear fluid, and not all reports have a common glaucoma-specific miRNA. We also conducted Human Practices with Prof. Ochiya, an expert in miRNA and exosome research, who told us that at present it is difficult to find a solid glaucoma biomarker miRNA in tear fluid even using meta-analysis, a type of bioinformatics technology. Therefore, instead of searching for candidate miRNAs in tear fluid, we decided to demonstrate POIROT using glaucoma biomarker miRNAs in aqueous humor, which have already been studied.

The concentration of miRNAs in the tear fluid was very low, estimated to be several hundred fM in total 4. We also found that in many cases, miRNAs that serve as biomarkers are expressed in both glaucoma patients and healthy subjects 5. We focused on three miRNAs (hsa-miR-10b-5p, hsa-miR-375, and hsa-miR-30b-5p) in the aqueous humor. These miRNAs have a 100:1 detection ratio between glaucoma patients and normal subjects, and POIROT aims to distinguish this difference to detect glaucoma 6.

Selection of Amplification and Quantitative Detection Module

In this section, we describe the following three steps in this year's project.

  1. Extensively investigate systems capable of amplifying and quantifying trace miRNAs
  2. List the requirements for our system
  3. Narrow down the list to a few systems that meet the requirements

Please refer to Engineering cycle for the steps to determine the reaction system to be used in the experiment, and Results for the steps to verify whether the constructed reaction system works.

1. Systems Capable of Amplifying and Quantifying Trace MiRNAs

Various systems have been developed to quantify trace amounts of biological samples.
In previous iGEM projects, for example, aptamers 7, 8, toehold-mediated strand displacement (TMSD) 9, Strand Displacement Amplification (SDA) 10, Rolling Circle Amplification (RCA) 10, 11, Loop-Mediated Isothermal Amplification (LAMP) 11 ,12, and others have been used. Each method is classified according to its characteristics.

  • Methods to amplify signals by biological/chemical systems
    • Methods to amplify nucleic acids from nucleic acids
      • SDA, RCA, LAMP, RPA 13, WGA, HDA, NASBA, TMA
    • Methods to amplify the presence of nucleic acids as signals other than nucleic acids
      • CRISPR-Cas, Aptamer, TMSD, HCR
  • Other methods
    • Femtoreactor 14, Graphene Oxide, Chemiluminescence, Lateral Flow Assay, Next Generation Sequencer, Nanopore

2. The Requirements for Our System

The system we are aiming for is a POC device to detect and quantify trace amounts of miRNA in tear fluid. The reaction system used in this system requires various constraints.
For example, the requirement of "no special equipment" means that RT-qPCR (Real-Time qPCR), a common method of nucleic acid amplification we first imagine, cannot be used. RT-qPCR is a robust method that combines polymerase and temperature change to efficiently amplify nucleic acids as the target product. However, RT-qPCR requires precise and frequent temperature changes, which is difficult to perform at home without a fluorometer or thermal cycler. In other words, a method different from qPCR is needed to detect the glaucoma biomarker miRNA in the home.
Similarly, we wrote down the conditions required for our system and decided to construct a reaction system with reference to these constraints.

The reaction should proceed at an isothermal temperature.
It is difficult to perform reactions that require raising and lowering the temperature in a home without a thermal cycler or heat block. Of course, there is room for consideration of creating a small device with thermal cycler functions, but since it is intended for home use, it should be a disposable device like a pregnancy test kit or a new coronavirus antigen test kit. Devices that require a power supply and an electronic circuit board are unlikely to be disposable and will be more expensive. Therefore, it is still necessary for the reaction to proceed at isothermal temperatures.

No special machinery is required to detect the signal.
Similar to constraint 1, but this is also a strong constraint. When creating a quantitation device for home use, fluorometers and capillary electrophoresis devices cannot be used. Although some iGEM teams created fluorometers and femtoreactors in their past projects, it is difficult to create a device at an affordable price for users. Therefore, it should not require special machinery to detect the signal.

MiRNA as input
Our biomarker of choice is miRNAs in tear fluid. Especially in tear fluid and aqueous humor, miRNAs are abundant in exosomes, and the exosomes must be destroyed during the reaction.

Quantification in the order of fM is possible.
Our selected biomarker contains only trace amounts in the tear fluid. Furthermore, when the sample is added to the reaction solution, the concentration of the biomarker in the reaction solution becomes even lower, so the method must be extremely sensitive.

Can be connected to other methods.
If the reaction can be linked to other methods, the requirement can be reduced. For example, if a reaction has high specificity but low amplification efficiency, constraint 4 can be overcome by combining it with a reaction that has low specificity but high amplification efficiency.
Furthermore, due to the nature of synthetic biology, it is useful to have systems that can be applied to other systems as modules. If the system can be applied to various systems, there is a possibility that it will be used widely in a new world, not limited to the target nucleic acid microdetection and quantification system such as this project.

3. systems that meet the requirements

1. Methods to Amplify Nucleic Acids from Nucleic Acids

This is a method to generate a large amount of single-stranded DNA by using DNA polymerase with strand displacement activity and nickase, which recognizes double-stranded DNA and inserts a nick into one of the strands. Various variants exist, and the target, amplicon, and reaction temperature can be changed for each way.

Main reaction mechanism:

  1. Target hybridizes with template nucleic acid.
  2. Polymerase elongates the 3' side of the target.
  3. Nickase inserts a nick.
  4. Polymerase elongates the target by removing the previous strand from the nicked site.

3-4 are repeated to generate a large number of strands on the 3' side of target.

No special machinery is required to detect the signal.
This method uses a cyclic DNA template and a polymerase with strand displacement activity (mainly Phi29 DNA polymerase) to generate very long repeat sequences. Various variants exist, and the target and reaction temperature can be changed for each.

Standard reaction mechanism:

  1. Target hybridizes with Padlock template to form a cyclic complex.
  2. T4 DNA ligase ligates the template to form a cyclic template.
  3. Primer hybridizes to the cyclic template.
  4. Polymerase with strand displacement activity extends around the cyclic template to generate a very long repeat sequence.

A complex method that uses a single template and 4 to 6 primers to amplify templates of varying lengths. It is applied in various detection systems and used in existing kits 16.

Standard reaction mechanism:

  1. Primers hybridize to the target DNA, and the polymerase extends.
  2. Another primer hybridizes to the newly generated template, and the polymerase extends.
  3. Repeated extension results in the formation of a dumbbell-shaped template.
  4. Another primer hybridizes to the loop region of the dumbbell-shaped template, and the polymerase extends.

This process repeats, producing amplified products (a smear) composed of concatenated dumbbell-shaped templates.

This is a method for amplifying dsDNA using recombinase.

  1. Primer and recombinase form a complex with the target dsDNA.
  2. Recombinase inserts the primer between the strands of the dsDNA.
  3. A polymerase with strand displacement activity extends from the primer, reforming the dsDNA.
Repeating steps 1-3 results in dsDNA amplification.

This is a method for genome amplification using a polymerase with strand displacement activity and random hexamers.

  1. Random hexamers hybridize to the genome.
  2. The polymerase extends from the random hexamers, causing branching through strand displacement activity.
  3. Random hexamers hybridize to the branches, and further extension leads to multiple layers of genome amplification.

This is a method to amplify dsDNA by unwinding dsDNA with helicase.

  1. DNA helicase acts on the target DNA to unwind the double strand.
  2. Primer hybridizes to the unraveled portion and polymerase elongates.
  3. New dsDNA is generated.

This is a method to generate dsDNA with complementary strands using RNA as the target.

  1. Reverse transcriptase acts on the target RNA to generate an RNA/DNA hybrid.
  2. The RNA side is degraded by RNase H.
  3. DNA polymerase activity of reverse transcriptase acts on the DNA side as a template to generate dsDNA.
  4. T7 RNA Polymerase acts with dsDNA as template, and target RNA is replicated. Steps 1-4 are repeated to amplify dsDNA.

This method targets RNA and generates dsDNA with the complementary strand.
It is almost the same as NASBA, but instead of RNase H, RNase H activity of Reverse Transcriptase is used.

There are many variants of these 8 methods, and the detection targets and reaction temperatures vary.
Methods that satisfy the requirement of "miRNA as input" are these two, including their variants.

  • SDA
  • RCA

The protocols of these two methods, including their variants, were investigated in detail. In RCA and other methods, even if annealing at 95 ℃ is required, if the amplification process is isothermal, it is often published as an "isothermal reaction". Therefore, we scrutinized whether any temperature change at all was necessary from the time the target was placed to the time the reaction was completed.

  • SDA

    • Prepare an enzyme mixture containing Bst 2.0 DNA Polymerase, Nt.BstNBI, Isothermal amplification buffer, and BSA, and a reaction substrate mixture containing template DNA, Isothermal amplification buffer, MgSO4, dNTP, dsGreen, and SSB, respectively. Dispense these solutions in 8-tube strips, and add target RNA. Incubated at 25 ℃ 15 sec → 50 ℃ 30 min, and measure fluorescence intensity every 10 sec.

    Prepare a reaction solution containing Bst 3.0 DNA Polymerase, nickase, NEB buffer, dNTP, Evagreen, and circular amplification template. Dispense this solution in 8-tube strips, and add target RNA. Incubated at 60 ℃ 60 min, and measure fluorescence intensity every 40 sec.

    Prepare a reaction solution containing hairpin probe, helper strand, Phi29 buffer, KCl, and DEPC water. Add target RNA to this solution, incubated at 90 ℃ 5 min → 37 ℃. Prepare a solution containing dNTP, RNase inhibitor, Nt.BbvCI, Phi29 DNA polymerase, primer, and MB probe. Add these solutions, and dispensed the mixture in 8-tube strips, incubat at 60 ℃ 60 min, and measure the fluorescence intensity every 20 sec.

  • RCA

    • Prepare a reaction solution containing open circle probe, gap probe, target miRNA, Tris・HCl (pH=8.3), KCl, NAD, MgCl2, Trition X-100, T. flavus DNA polymerase, T. thermophilus DNA ligase, and Ampligase DNA ligase, and dispensed in 8-tube strips. Incubate at 52 ℃ 1 hour. Prepare a solution containing circularized probes, rolling-circle primer, MgCl2, acetylated BSA, dNTP, Phage T4 gene-32 protein, and Phage Phi29 DNA polymerase. Add these solutions, and incubate the mixture at 31 ℃ 30 min and measure the concentration of ssDNA produced.

    Prepare 50 -phosphorylated circular probe precursors. Prepare a reaction solution containing TWJ probes and PG-RCA buffer. Dispense this solution in 8-tube strips, and add target RNA. Incubated at 37 ℃ 90 min → 80 ℃ 5 min. Prepare an enzyme solution containing dNTP, circular probe, Vent(exo-)DNA polymerase, and Nb.BsmI. Incubate at 60 ℃ 90 min, and measure fluorescence intensity every 20 sec.

    Prepare a reaction solution containing loop open dunbell DNA with a phosphate group at the 5'end, miRNA, and SplintR ligase buffer. Incubate at 94 ℃ 5 min → 10 ℃. Add SplintR® ligase and incubate at 25 ℃ 16 hour → 65 ℃ 20 min → 10 ℃.
    Prepare a solution containing exonuclease III. Add this to the previous reaction solution and incubated at 37 ℃ 20 min → 10 ℃. Add urea solution and incubate at 95 ℃ 5 min → 10 ℃. Conduct capillary electrophoresis with 12% polyacrylamide gel, cut out the desired band from the gel using a scalpel, add PBS, and stir calmly for 24 hours. Centrifuge at 4 ℃, 12000 rpm, 5 min using Vivaspin® 500 centrifugal filter, determine the concentration by Nanodrop, and stored at 4 ℃.
    Prepare a reaction solution containing the obtained circular dumbbell DNA, and Phi29 DNA polymerase buffer. Dispense this solution in 8-tube strips, and add target RNA. Incubate at 94 ℃ 5 min → 10 ℃. Add Phi29 DNA polymerase and dNTP and incubate at 30 ℃ 40 min → 65 ℃ 10 min → 10 ℃.
    Prepare a solution containing PstI endonuclease and NEBuffer r3.1, and add it to the previous reaction solution. Incubate at 37 ℃ 10 min → 80 ℃ 20 min. Prepare a PBS solution containing HD probes, and add it to the previous reaction solution. Incubate at 94 ℃ 5 min → 10 ℃, and measure 496 nm fluorescence intensity.

    Prepare a reaction solution containing DNA solution (L1), 5'-phosphorylated DNA solution (T1A), 50% w/v PEG 6000 aqueous solution, T4 DNA ligase, Phi29 buffer, and NaCl. Incubated at 37 ℃ 1 hour, add BPB solution, and quench. Incubate at 94 ℃ 5 min and purified with 10% PAGE gel. Prepare other templates in the same way.
    Prepare a reaction solution containing template cT1c, template cT2, primer p2, dNTP, Phi29 DNA Polymerase, Phi29 Buffer, and BSA. Dispense the solution in 8-tube strips and add target RNA. Incubate at 37 ℃ 2 hours, add PBS solution, and quench. Dispense this solution in 8-tube strips, add fluorophore (ThT-HE or SYBR Gold) in PBS solution, and incubate at room temperature for 30 min. Measure the 450-600 nm fluorescence intensity.

    From the above, only SDA satisfies the requirement that the reaction proceeds at isothermal temperatures.
    Furthermore, since reverse transcription is a slow reaction, it is also important for POC devices not to require reverse transcription: SDA does not require a reverse transcription reaction and has an advantage in this respect.
    Therefore, we decided to consider SDA as a candidate for the amplification and quantification module.

    2. Methods to Amplify the Presence of Nucleic Acids as Signals Other than Nucleic Acids

    The CRISPR-Cas system is essentially a system maintained by bacteria as an immune response to viral infection, which works as follows. First, bacteria infected with a virus incorporate the viral DNA sequence into the CRISPR region of their own genome. Next, the CRISPR site is transcribed and processed to produce a crRNA of about 30 bases. Finally, a group of Cas proteins form a complex with the crRNA and defend themselves against the virus by cutting up the viral dsDNA corresponding to the incorporated sequence.
    The CRISPR-Cas system, as it is well known, is widely used as a genome editing tool and was selected for the 2020 Nobel Prize in Chemistry 25. It is worth mentioning that the ability to recognize nucleic acids in a sequence-specific manner and fuse them with transcriptional activators and repressors can provide gene-switching functions. CRISPR-Cas has been used many times in past iGEM projects, and various CRISPR-Cas systems have been registered as Parts.

    Generally, CRISPR-Cas is used to introduce mutant genes or as a gene switch, but we focused on the collateral activity of some CRISPR-Cas systems, which means that when CRISPR-Cas recognizes a complementary sequence to crRNA, it cleaves the complementary sequence (cis activity). When CRISPR-Cas recognizes a complementary sequence to crRNA, in addition to cis activity, it also non-specifically cleaves nucleic acids in the vicinity (trans activity, collateral activity). Once the collateral activity occurs, approximately 1000 nucleic acids are cleaved, making this activity useful for sequence-specific signal amplification.
    CRISPR-Cas9, the most famous Cas protein, has no collateral activity, while Cas3, Cas12a, Cas13, Cas14 have collateral activity.

    Cas3 is a CRISPR-associated protein that functions with the Cascade-crRNA complex. It has a 27-nucleotide spacer sequence, recognizes dsDNA, works well at 37℃, and has ssDNase collateral activity. A characteristic property of Cas3 is its "shredder" function, which randomly cleaves in front of the recognition sequence after cis activity. This property may enable large-scale gene editing. One of the strengths of Cas3 in miRNA detection systems is its high specificity for sequence recognition due to its long spacer sequence compared to other Cas proteins. Currently, Cas3 is being intensively studied in the Mashimo Laboratory of The Institute of Medical Science, The University of Tokyo 44, and is being commercialized by C4U, a venture company originating from the Mashimo Laboratory.

    Cas12a is a CRISPR protein with a 24-nucleotide spacer sequence that recognizes dsDNA.
    It works well at 37℃ and shows DNase activity after activation.
    The main advantage of Cas12a is that it does not require tracrRNA or RNase Ⅲ. However, its weakness is that it requires incubation with crRNA at least 24 hours before use. Another potential weakness is the relatively short recognition sequence.

    Cas13 is a CRISPR protein with a spacer sequence of 22-28 bases (in the case of Cas13a) that recognizes ssRNA. It works well at 37-39℃ and shows indiscriminate RNase flanking activity after activation. The strength of Cas13 is that it is composed of a single effector protein and gRNA, making it easy and fast to design. On the other hand, its weaknesses are the possibility of off-target mutations and the lack of DNase flanking activity.

    Cas14 (also known as Cas12f) is a CRISPR protein with a 27-29 base spacer sequence that recognizes ssDNA. It works well at 50℃ and shows indiscriminate ssDNase activity after activation. Cas14's strength is its ability to recognize SNPs not detected by Cas12 due to its higher specificity than Cas9, Cas12, and Cas13. Another advantage is its small protein size compared to other Cas proteins. However, its weakness is that it is a relatively newly discovered protein and has not been well studied. In addition, its relatively high activity temperature of 50℃ is also a limitation in its application.

    The aptamer is a synthetic DNA/RNA that binds specifically to a particular target molecule. It can bind to a wide variety of target molecules and can also bind specifically to nucleic acids. The antisense strand of the aptamer can be detected by modifying it with a fluorescent substance 37. In the past, it was used in IISER-TVM 2023 7 and IIT-ROORKEE 2023 8 to detect miRNAs and proteins in the body: it can be made in vitro and has very low detection limits, although it is highly stable and depends on the detection method. It can be combined with logic circuits such as 'and gate' and shows a response to multiple inputs 38. This means that multiple types of miRNAs can be detected simultaneously as input signals. However, if disease-specific miRNAs are present in both disease-positive and disease-negative individuals, or if there are large differences in concentration among miRNAs, the output result would be inappropriate because it depends on a subset of the miRNA population that one wishes to detect. Another drawback of this method is that it is difficult to make it quantitative enough, while it is suitable for binary decision.

    TMSD (Toehold-Mediated Strand Displacement) is a non-enzymatic method of replacing target nucleic acids with nucleic acids of a different sequence 39.

    1. Hybridize the target DNA or RNA with a template so that a portion of the target DNA or RNA becomes a duplex.
    2. Part of the duplex created in step 1 is a single strand called a toehold, and the new DNA or RNA molecule binds to this short overhang
    3. After binding to the toehold region, the new DNA or RNA molecule moves along the other complementary regions of the original duplex, replacing the original strand.
    4. When the new molecule completely displaces the target nucleic acid, the target nucleic acid dissociates from the duplex.

    The repetition of steps 1-4 produces the amplification product.

    HCR is a method to amplify dsDNA containing many nicks with Enzyme free from miRNA or ssDNA as input. 3 types of templates with hairpin structure are required and the reaction is performed at 37 ℃.
    Standard reaction mechanism:

    1. miRNAs hybridize to hairpin1 to form a complex with extended legs.
    2. Hairpin2 hybridizes to the foot of the complex, forming a complex with extended legs.
    3. Hairpin3 hybridizes to the foot of the complex, forming a complex with extended legs.
    4. Hairpins 2 and 3 hybridize repeatedly to the foot of the complex, and long dsDNA is generated.

    None of these have not been solved these problems:

    • The possibility of quantification on the order of fM
    • No special machinery is needed to detect the signal

    Therefore, from the viewpoint of "being able to connect with other methods", candidates for the amplification and quantification module are as follows.

    • CRISPR-Cas
      • Cas12a
      • Cas3
    • HCR

    3. Other Methods

    Femtoreactor, which has been used in iGEM in the past 14, 40, is a revolutionary technology that can distinguish the presence of a single molecule. External devices such as high-performance cameras and smartphones are required for detection.

    Graphene oxide's affinity for hydrophobic biomolecules can be used to detect many biomolecules, including single-stranded oligonucleotides, and methods have been developed to specifically detect miRNAs 41. By using a complex of nucleic acid peptides bound with fluorescent molecules and graphene oxide, a system can be constructed that generates fluorescence when the complementary miRNA binds to the nucleic acid peptides. This allows miRNAs to be specifically detected and quantified. However, a device for measuring weak fluorescence is required, which is considered inappropriate in this case.

    Nucleic acids are not directly substrates for chemiluminescence. Since only a limited number of targets can be detected by chemiluminescence, a step is necessary to change the presence of nucleic acids into another signal that can be detected by chemiluminescence.
    Various substances can be used as targets for chemiluminescence detection, as follows.

    • Luminol-based
      • H2O2
      • Heavy metal ions such as Fe3+ and Cu2+, etc.
      • Peroxidase
      • Hemin
      • Fluorescent substances
      • etc.
    • Adamantyl-1,2-dioxetane derivatives
      • Alkaline phosphatase
      • β-D-galactosidase
    • Ruthenium complexes/electron donors
    • Dioxetane Derivatives
    • PO-CL (Peroxyoxalate Chemiluminescence)

    Although these methods have been devised to increase luminescence intensity, they are far from visual confirmation. In addition, many of these methods produce strong luminescence that lasts only for a short period of time because the system is designed for the use of special equipment such as photomultiplier tubes. Therefore, chemiluminescence was considered unsuitable for POC devices.

    A simple test method, well-known for pregnancy test kits and other products, can be designed to form a visible band when certain molecules are present in the specimen: LFA is quick, easy to use, and does not require specialized equipment, making it widely used as a POC device.

    Next-generation sequencers are an innovative technology that can decode large numbers of nucleic acids in parallel with high sensitivity. However, it is not suitable for use as a POC device because it requires advanced facilities and time to obtain results.

    A technique for sequencing nucleic acids by measuring the change in current as they pass through a nanopore, and portable machines such as MinION 42 have been developed. However, this technology has a relatively high error rate and is still in the research phase, so its direct use as a POC device is still considered difficult.

    • Reaction proceeds at isothermal temperature
    • Quantification on the order of fM is possible
    • No special machinery is required to detect the signal.
    • MiRNA is used as input.

    We decided to consider LFA as a candidate for the amplification and quantification module from the viewpoint that it can be connected to other methods.

    Systems That Meet the Requirements

    We considered

    • SDA
    • HCR
    • CRISPR-Cas
    • LFA

    as a candidate for the amplification and quantification module.

    Design of POIROT

    Click here for our process to consider SDA, HCR, CRISPR-Cas, and LFA to decide reaction system for Wet Lab experiments Engineering cycle
    Click here for our testing process for functionality of system in POIROT Wet

    Here, we describe the summary of the system for POIROT.

    1. Users collect their tear fluid using Schirmer strips.
    2. Glaucoma biomarker miRNA in tear fluid is specifically amplified in the form of dsDNA by TWJ-SDA, Multistep-SDA, and ds-amplification.
    3. Cas3 recognizes the amplified dsDNA and cleaves the surrounding ssDNA.
    4. If the user is positive for glaucoma, both the Test line and Control line on the detector turns red, while if negative, the Control line turns red.

    In this way, POIROT can detect glaucoma at home with its ease to use.

    For Social Implementation

    Safety and Ethics

    In developing the device, we have carefully considered the Safety and Ethics matters so that the device can be used at home. Japan has ratified the Cartagena Protocol 43. The Cartagena Protocol has strict regulations regarding the handling of GMOs (genetically modified cells). Therefore, instead of using cells, we developed a protein-based system to contribute to preventing the spread of GMOs. Since the user only collects tear fluid, the system has very low invasiveness to the human body. Furthermore, to prevent damage to the eye during collection. To enable users to easily interpret results, we simplified the reading process by utilizing color development on the calibration curve of the LFA.

    To verify that our system is truly safe and ethical, we discussed with Prof. Muto, from the field of public policy research, and the following were pointed out:

    • It does not violate the Cartagena Protocol for now
    • It might violate the Cartagena Protocol when actually commercialized.
    • The need to consider the dual-use of the product (use for purposes other than intended and potential harmful effects). We discussed the dual-use of the product within the team and concluded that there are no issues. However, we further deliberated on this topic during discussions with the general public. Check Education for details.

    To demonstrate the system, we decided to use actual tear fluid. For this, we need to submit ethical review documents to the ethics committee of the institution (The Institute of Medical Science, The University of Tokyo) to get their approval. When doing so, we primarily focused on the following:

    • Obtaining informed consent from patients and confirming their agreement.
    • Storing collected personal information, such as the patient's medical history of ophthalmic diseases, in accordance with internal regulations.
    • Responding to patients' requests to withdraw consent after tear collection.

    Further information on the clinical trial is written in the Clinical Test section.
    In addition, see Safety & Security for more details on the safety of the project.

    Clinical Trials

    To demonstrate POIROT's amplification and quantification system, we need to use human tear fluid. As mentioned above, approval from the ethics review committee is necessary. With the cooperation of our affiliated laboratory and the Ethics Review Support Office, we prepared the necessary application documents. Initially, we considered tear fluid collection to be completely non-invasive, but as a precaution, we decided to classify it as minimally invasive. In addition to the potential risks associated with such experiments, we also had to consider the possibility that clinical trial participants might experience harassment from the experimenters, ensuring that various precautions were taken.

    The required application process is as follows:

    1. Preparation of Required Documents: We submitted six types of documents, including the ethics review application form, research plan, flowchart (showing the flow of samples and information, and the relationship with other institutions), explanation documents, consent forms, consent withdrawal forms required for informed consent, a conflict-of-interest self-declaration form, a questionnaire, and posters for recruitment.
    2. Completion of Ethics Training: All members involved in the research are required to complete the ethics training specified by The Institute of Medical Science, The University of Tokyo. The training must be completed before the application is approved and must be retaken after a few years.
    3. Responding to Preliminary Opinions: The research content and methods outlined in the research plan, as well as the consent acquisition and withdrawal procedures, were reviewed by the ethics committee members before the committee meeting. Preliminary feedback was provided, and we carefully reviewed whether the application content was appropriate while responding to the preliminary opinions.
    4. Attendance at the Ethics Review Committee Meeting: The applicant is required to attend the ethics review committee meeting, answer questions from committee members, and respond appropriately.
    5. Approval of the Application: If the application is approved by the ethics review committee, the process is finalized, and the ethics application process is completed.

    We would like to express our gratitude to the members of our affiliated laboratory and the ethics review support office who assisted with the ethics application.

    The process following the approval of the ethics application is as follows:

    1. Recruitment of Participants: We will recruit individuals willing to provide tear fluid using posters and other materials.
    2. Consent of Participants: The research content will be explained to participants, and they will provide consent for tear fluid collection. Care must be taken to ensure that the explanation is clear enough for anyone to understand the research content. Additionally, we will avoid asking participants again if they initially decline, and special care will be taken to avoid coercive consent based on positional authority.
    3. Tear Fluid Collection: Tear fluid will be collected from participants who have given consent.
    4. Data Analysis: We will use the collected tear fluid to demonstrate the amplification and quantification system of POIROT. The results obtained will not be communicated in a way that causes any psychological burden to the participants. If a participant withdraws their consent, their tear fluid and data will not be used.
    5. Report: Before the predetermined research period ends, we will report the implementation details to the ethics review committee.

    The process after the approval is scheduled for future implementations.

    Other than Glaucoma

    At first, we thought of POIROT as a system contributing to the early detection of glaucoma. However, the high flexibility of the amplification system suggested that it could be applied to the detection of other diseases as well. By simply modifying the sequences around the target recognition region, it is possible to detect different miRNAs. In other words, POIROT could detect disease-specific biomarker miRNAs for conditions other than glaucoma. POIROT holds the potential to detect a variety of diseases.

    hogehoge

    Figure 4.

    How to Realize

    POIROT's amplification system can be boldly divided into two parts: the target recognition section and everything that follows. Originally, the target recognition section uses a template with a sequence complementary to the glaucoma biomarker miRNA.
    By designing these two templates and helper DNAs to hybridize with specific miRNAs, we enabled sequence-specific amplification of miRNAs. Beyond the target recognition region, the system remains unaffected by the miRNA sequence, eliminating the need for further system changes. This highlights POIROT's flexibility. In the Dry Lab, we enabled the design of appropriate sequences for different miRNAs, facilitating the application of our system to other diseases. Details are available in the Model_Sequenece Design.

    POIROT has the potential to be applied beyond early detection. It is capable of quantification using fluorescence, making it possible to track the progression of diseases. This approach requires an assumption that there is a correlation, or preferably a causal relationship, between miRNA expression levels and disease progression. By continuously quantifying miRNA concentrations with POIROT and tracking changes, we can monitor disease progression. This technology could be used for diseases like glaucoma, which progresses with few early symptoms, as well as to track the effectiveness of treatments through miRNA concentration monitoring.

    Future Plan

    Improvement of POIROT

    POIROT still has the potential to evolve:

    Through repeated experiments, we have successfully developed a detection system with both high selectivity and sensitivity. However, to fully complete POIROT, further experiments from both Wet Lab and Dry Lab approaches are essential. Our amplification system achieved the target detection of 1 fM, which was accomplished under ideal conditions, not yet reflective of reality. The most critical challenge is to verify whether we can amplify target miRNA present in trace amounts within human tear fluid, a highly complex environment filled with contaminants. First, we will amplify miRNA known to be present in relatively high quantities in the tear fluid of the general population, confirming that our system works effectively in human tear fluid. Click here for further approach experimentation from Wet Lab and Dry Lab.
    As discussed in Human Practices, POIROT holds potential beyond Japan, especially in medically underserved regions or areas with low healthcare standards. To make it accessible to anyone, anywhere, we must ensure that results are displayed in a simple, easy-to-understand format. This requires optimizing the LFA strip for POIROT, pushing sensitivity and specificity to their absolute limits.

    We must continue refining POIROT. Our mission is to ensure this detection device gains widespread adoption, contributing to the early detection and monitoring of various diseases, not limited to glaucoma. To achieve this ultimate goal, we must not stop our efforts.

    Commercialization of POIROT

    To ensure POIROT's proper use in society and its commercial sustainability, we aim to rapidly implement it. We will devise business "scenarios" to determine whether POIROT can withstand the pressures of entrepreneurship, exploring the possibilities for its social deployment.

    The first step toward product sales is to understand the characteristics of both the demand and supply sides while recognizing the strengths and weaknesses of our assets. On the demand side, we investigate the current market, its growth potential, competitors, and the value of the product to customers. On the supply side, we assess our production and supply capabilities as well as the value chain. Based on this, we outline a roadmap for the business growth strategy. We set business milestone goals over time and consider how the company should operate within society to achieve these goals, while also planning for the long-term impact of the business and how to address it.
    For more details, see Entrepreneurship.

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