Diagnostics Via Disc

Download Detailed Research Proposal click here.

Problem: COVID testing is slow, expensive, does not scale well

• India is a country having a population of over 1.35 billion, the testing rates for COVID in India is hardly any spectacle because of the high cost of testing, lack of indigenous test kits due to slow manufacturing, the skill of end-users, unavailability of costly testing apparatus like RT-PCR etc.
• To devise a method to diagnose COVID 19 which uses easily available electronic equipment like CD DVD players, which employs cost-effective sample preparation methods to provide fast and reliable results.

Our Solution: test many samples at a very small cost

• Our software uses CD/DVD disc reader technology for quantitative analysis of bio-assays prepared on CD/DVD discs to detect COVID 19, can perform antibody/antigen tests, colourimetry tests etc rapidly and with high specificity and sensitivity but most important of all at relatively negligible costs.
• The cost or our method is very low because it uses hardware that is pervasively available worldwide: any computer that can run Linux, any CD/DVD player designer for a computer, possibly needing a cheap adapter to connect both. It even runs on a Raspberry Pi model 1 through USB.
• A method to prepare the surface of the disc for preparing the assay on CD/DVD surface to test for COVID 19 using a special protein (a short chain of amino acids connected by peptide bonds). Although what matters the most is the diagnostics technology rather than which biological test we perform on it which ensures it's usability and necessity in the future markets as well.

Our proposed solution involves surface modification of the polycarbonate layer of the optical disc (CD/DVD) then an assay similar to that of micro ELISA sandwich format is developed to detect SARS COV-2 and characterization of the assay is done by CD/DVD disc reader technology. The detection and capture molecule chosen for this is a small peptide.

Disclaimer: We are still testing the efficiency of the protein and waitingfor peerreviewed and verified results. When this phase is complete we will be sharing all the test results.Initially we have developed the software for COVID, butwe can perform other bio assay tests and many other diagnostic tests as well which will serve as a long term and low cost solution in developing countries.

The Project at a Glance

The Setup

List of items used:

• Raspberry Pi with power supply
• CD (or DVD/BluRay) reader
• USB to IDE/SATA adapter (in this example the CD reader is IDE) with its power supply and cable.
• Display monitor, power cable, HDMI cable
• Keyboard, mouse
• Power distributing cable

Our Team

We are a team comprised of:

• Gourav Samal (Undergraduate in Materials Engineering, NIT Rourkela, India) as project leader.
• Stephane Gourichon (Sorbonne Université, Paris, France) as a software engineer and project galvanizer. Engineer degree from École Polytechnique (ParisTech), Ph.D. in computer science from Sorbonne Université, extensive software experience on industrial/embedded systems, including transportation, defense, security and health.
• Professor Ashwin Gopinath (MIT, USA) as our biological expert and advisor. PhD in electrical engineering from Boston University, a research scientist in the Department of Bioengineering at the California Institute of Technology. Awarded Robert Dirks Molecular Programming Prize.
• Vamsi Krishna Gotti (Awalk, Founder & CEO). Undergraduate in Electronics & Instrumentation Engineering, NIT Rourkela, IndiaExperience in System Design, Firmware development, & Web App development.
• Guna Sai (Undergraduate in Materials Engineering, NIT Rourkela, India) acting as a team support member.
• Some of us found each other at HEC/Polytechnique/SciencesPo, Paris COVID-19 hackathon. We all are working remotely from our homes because of lockdown.

First things First:Lets get some basics done which is very crucial for understanding the project, lets understand how a DVD drive works. Please visit this link for detailed explanation, core concepts and some bonus materials for geeks.

List of References

Considering the complexity of the topic and the interdisciplinary nature, people currently in the field are already familiar with ELISA tests but not with the CD/DVD drive based diagnostics. So we are providing a link which contains a list of scientific articles and supplementary material which will further help them in understanding and interpreting the obtained results. Use this link for downloading all the supplementary documents used in this project which is present in a single folder.

The Software

We have developed a GUI for performing the analysis of bioassays.

We wrote a convenient Graphical User Interface (GUI) targetting the cheap computer Raspberry Pi and can work also on Linux desktop machines, to leverage the many existing computers.

We tested it successfully on regular PCs running Ubuntu 20.04 and 18.04 and ArchLinux, and on a Raspberry Pi model 1 running Raspberry Pi OS 10 (Raspbian, based on Debian 10 buster).

Here is how the GUI looks like:

The GUI is built upon Python, NumPy, Qt, pyqtgrah and libcdio.

If you have a computer running Linux and ability to install the software as root (scripts are provided for Debian and Ubuntu), instructions are in the Github repository.

Then you can run the GUI with this typical workflow:

• open GUI
• select the CD-Rom drive you wish to use (if only one it is already selected)
• Convenience buttons "eject" and "close tray" are provided should you have a drive that has no physical button
• Insert the CD that holds the samples you have prepared and close the tray.
• Click on "start analysis"
• The analysis will last for a few minutes and a graph will appear in real time.

This animated GIF show progression:

Please refer to the Github page readme file for more details, including installation steps. We also have provided bash scripts for easier installation.

Some drives may take more or less time to perform the analysis. Some drives may get stuck for a long time. You can press "Stop" at any time to cancel the analysis and keep the partial graph.

The graph at the bottom resizes with the window and is an interactive pyqtgraph where you can zoom. You can export CSV data for reference.

The position and height of peaks are used as the basis to discriminate between samples and provide the diagnostics.

Please check this document for performing the test and how to recognise and interpret the results.

Error based quantitation of Bio-assays

Compact discs (CDs) can be repurposed to be used as rapid, low-cost, high-throughput screening platforms for running bioassays without modification of the detection hardware (standard optical drive). We have developed software which identifies erroneous sectors by locating the error position in the form of C1/PIE and C2/PIF errors. The number of erroneous sectors increases asa function of the concentration of the analyte.

This method can be used to performquantitative analysis of ELISA and many other types of quantitative analysis, colourimetry as well. Now let’s understand what is anELISA.

ELISA

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying peptides, proteins, antibodies, and hormones. In ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. An antigen-antibody complex is a molecule formed from the binding of multiple antigens to antibodies. The bound antigen and antibody act as a unitary object.

Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction. Enzyme-linked immunosorbent assays (ELISA) principles are very similar to other immunoassay technologies. ELISAs rely on specific antibodies to bind the target antigen, and a detection system to indicate the presence and quantity of antigen-binding. In order to maximize the sensitivity and precision of the assay, the plate must be carefully coated with high-affinity antibodies. (“ELISA Fundamental Principle, How It Works.” [Online]. Available: https://www.bosterbio.com/protocol-and-troubleshooting/elisa-principle.)

Now, theseare the biological details of the ELISA test. For quantitative analysis, a device called the ELISA reader is used.For an indepth understanding of the ELISA testsand how an ELISA reader works, refer this link.Now we will understand how oursoftware works and how are we going to use a CD/DVD to mimic this device's features and produce accurate results.

IMPORTANT NOTENow what we are going to do is we are exploiting the features of a CD/DVD drive to perform the quantitative analysis using similar principles as that of the ELISA reader i.e. measuring the absorptance of light by the assay. Now, we directly don’t have access to the raw signals from the sensor so we have found an indirect way to measure the concentration of the analyte. This will be explained in the upcoming sections.

Errors in CD/DVD discs

Now let’s understand what C1/PIE and C2/PIF errors are and how they are used for quantitative analysis.

1. C1 errors: These are the lowest level bit errors and are usually measured as errors per second. This block error rate or (BLER) is an indication of the quality of the disc.

2. C2 errors: These can be caused by poor master CD burning, poor mastering media and also scratches, marks and scuffs to the disc. C2 errors are read errors and not always a physical defect of the disc.

3. PIE (DVD only): Parity Inner errors (PIE) is the number of parity inner rows with errors. If a PIE cannot be corrected by the drive it counts as a PIF. If a PIF cannot be corrected by the drive it counts as a POE.

4. POE (DVD only): Parity Outer errors (POE) are the number of uncorrectable parity outer columns in an ECC block.

(“CD Duplication, DVD Duplication, CD Replication UK Services » Blog Archive » CD duplication C1, C2 and CU disc errors.” [Online]. Available: http://www.testa-rossa.com/cd-duplication-c1-c2-and-cu-disc-errors/.)

NOTE: Formuch more detailed explanation of the errors and how they are generated, error correction methodologyetc.please visit this link.For DVD drives, the PIE and PIF are generated based on a different algorithm. Please visit the link below for more details.Standard ECMA 267 (http://www.ecma-international.org), p. 26–30

Optical Phenomenon

Now let’s understand how all of this tech jargon fits into our project:

A conventional optical disk drive for reading DVDs and CDs contains all needed components to perform quantitative chemical and biological analysis. The drive has two lasers, 650 and 780 nm, to read DVDs and CDs, respectively, a Si photodiode detector, and a sophisticated laser tracking system to scan across the disk surface. In the developed lab-on-DVD sensor system, an analog signal from the photodiode is extracted before it is digitized during the reading of the digital content from an optical disk and brought into a data acquisition program. This signal is used for quantitative detection of changes of optical properties of chemical or biological assays deposited on the read surface of the optical disk.

In the absence of an assay, laser light is transmitted through the surface of the optical disk, reflected from the disk’s reflective data layer, and returned to the photodiode detector. When an assay is applied onto the read side of the CD or DVD disk, the laser light travels through the sensor film twice as shown in the figure above. Upon interactions of the sensor film with chemical or biological species, optical properties of the sensor film vary causing the change in the amount of light detected by the photodiode detector of the laser pickup head and allowing for quantitation of assay response.

The optical system of a conventional CD/DVD drive focuses the laser light onto a reflective layer inside the disk to a spot of ∼1 µm and provides polarization and phase control of the light that reaches the detector. In reading digital data from a disk, these features are important for the rejection of ambient light and light produced by scratches and other imperfections on the disk surface. In the current application of optical disk drives, these features provide an opportunity for the chemical and biological quantification based on a variety of optical phenomena that can be produced in the assay (see above Figure B). (R. A. Potyrailo, W. G. Morris, A. M. Leach, T. M. Sivavec, M. B. Wisnudel, and S. Boyette, “Analog signal acquisition from computer optical disk drives for quantitative chemical sensing, ” Anal. Chem., vol. 78, no. 16, pp. 5893–5899, 2006.)

Principle

This is the most important part, the basis of our project, how it works.

Now, for the quantitative analysis (or one can call it as Multiplex biological screening) we have developed a software which will perform the quantitative analysis based on the error counts which are produced because of the various optical phenomena mentioned above. As these phenomena depend upon various factors, we must control some of them to measure the desired parameters concentration of the analyte, presence and absence of proteins etc. So, any irregularities in the surface of the disc will produce various effects. These effects will present themselves in the form of error counts but for this, to work we need to understand that objects of ∼200 nm size (> λ/4) are needed to induce significant disruption of the laser light reflection (to create “readable” digital signals) in a standard optical drive.

Biological macromolecules (typically a few nanometers) are too small to be detected; therefore, the molecular binding signals must be amplified. What we mean by this is that using readily available detection molecules we can amplify the signals by producing molecules which are large enough to block/scatter the laser light producing read errors detectable by the optical drive. At saturated concentrations, the binding spots can be observed with the naked eye. For more details please refer ELISA substrate and Standard ELISA procedure.

Note that we use positive and negative controls in assays so that we can check for false positives, validity of the assay and optimize the assay procedure.

We marked the surface of the disk with a dye, with varying concentration. The software will scan the disc and generate a plot based on error counts on the y-axis and its logical location(called as sector number)/playtime on the x-axis. An example is given below.

IMPORTANT NOTE: We can standardize the test results by using samples/analyte whose concentrations are known to plot calibration curves. Then scan the experimental sample and fit the data according to the calibration curve and find the concentration of the analyte.

Using our software one can perform ELISA tests and many more experiments on the surface of the discs itself and with high sensitivity. The location where the reaction/binding event has happened on a CD (if it significantly disrupts the laser reading process) can be identified by using the formulae given below.

As shown in Figure below, the radius of the programmable area of a typical 700-MB CD-R that contains 79.7 min of audio data is 58 mm, and 25 mm is the radius of the centre section. This programmable area is our experimental region. The assay is to be developed in this part only otherwise it will lead to an error while reading the disc.

For a DVD, the same formula can be used by replacing the constants with those according to the specifications of DVD. The formula can also be written in a different format using logical position instead of playtime.

For a standard size, single-sided DVD, the total data storage capacity is 4.7 GB and the data recorded in the area between 24 mm and 58 mm in terms of the radius. Both equation 1 and 2 are necessary because we can transform the error distribution plots based on the corresponding physical location i.e. the radial distance of the sector from the centre of the disc. This eliminates the broadening of the data as there are more sectors on the outside of the disc than on the inside.

Bio-assay on an optical disc

There are different kinds of bio-assays that can be performed on the surface of a CD/DVD. For this project, we just consider making an ELISA on the surface of the optical discs (CD, DVD etc.). The surface of the CD, DVD’s is made up of polycarbonate material which makes them suitable for performing ELISA tests as extensive research has been done to optimize the test parameters. However, we must note that several other tests like antibody/antigen-based tests for virus detection, checking water contamination levels and many more tests can be performed using the same principle and optical phenomenon described earlier. The possibilities are limitless.

Specifically for detection of COVID 19, we have found a suitable protein which will act as a detection molecule/antigen/antibody. We need extensive clinical trials before we can be 100% sure about the actual figures and can optimize the testing parameters and quantity of the reagents. Till then we won't be able to provide the exact protein as we haven't yet received verified results. As far as past research goes, this method of using error based analysis can detect with sensitivity more than that of 80%.

Please find the detailed steps for preparing the ELISA assay on an optical disc in this link. Do exercise caution as this topic is very sensitive as it's related to the diagnosis of infectious diseases.Although one can perform very simple experiments with this software as well.

Frequently Asked Questions

Q. How can someone make any meaningful measurement when there are so many drives, most with so flakey firmware, and which aren't even meant to be measurement devices?

A. This is proof of concept of our idea. We are working to get better sensitivity and specificity by using custom hardware i.e CD/DVD drives with minor modifications and some extra hardware to standardize the results. We are also planning to provide discs with known properties and which will be suitable to run these type of tests.

Q. What about runtime errors like the discs get stuck, the drive may vary, doesn't get recognised by the drive etc?

A. This can be dealt with using recommended hardware which we have mentioned in Bill of materials as well as in particular documents earlier. These can be solved by other means; case to case basis.

Conclusion:

• Our software helps in ramping up testing for COVID 19 and other bio-assays at very low initial investment costs and per sample reagent costs.
• It provides a platform for technical/non-technical users as well to perform the quantitative analysis of bioassays with ease.

Lessons learned

• No matter where people are from, wherever they are from, even from their homes can come together to build something out of cheap electronics which is truly amazing.
• Consumer electronics like CD/DVD are modern engineering marvels. However, how they work, access to their complete functions, are not available openly and are not well documented.

Future plans

• We are building another prototype which uses a DVD drive with some additional hardware to get raw data out of the drive and provide better sensitivity while performing assays.
• Making a standalone diagnostic system in a point-of-care setting providing cheap and high throughput diagnostics.