Description

Introduction

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Inspired by the increased cases of patients suffering from Non-Small-Cell-lung-Cancer in combination with the poor prognosis and the low survival rates, due to the inability to detect the disease in the early stages, our team decided to design DIAS. DIAS concerns a low-cost, non-invasive, effective diagnostic device for the early detection of Non-Small-Cell-Lung-Cancer, utilizing innovative technologies and methodologies, blood as sample and miRNAs as biomarkers. The DIAS project refers to the development of an innovative molecular diagnostic platform for non-small cell lung cancer (NSCLC), combining Cas13 technology with microfluidic methodology and imaging approaches.

miRNAs

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MicroRNAs (miRNAs) are a group of small non-coding RNAs of 17-25 nucleotides in length that are conserved across species. They were first discovered in Caenorhabditis elegans at the beginning of the 1990s. miRNAs are expressed in different tissues and cell types and are involved in regulating a range of developmental and physiological processes. Deregulated expression of these small RNAs have a significant impact on development of diseases including cancer. Over 1900 miRNAs have been reported that have critical regulatory functions and are involved in virtually all physiological processes, such as cellular development, proliferation, and differentiation metabolism and homeostasis (Iorio et al., 2012).

Many human miRNAs appear to control important processes that play vital roles in the onset, progression, and metastasis of cancer like:

• cell proliferation
• cell adhesion
• apoptosis
• angiogenesis

MiRNAs have shown high stability in formalin-fixed, paraffin-embedded tissues from lung cancer in human plasma, raising the possibility that miRNA expression analysis from archived tissue samples and body fluids including blood will be useful for characterizing disease states. The vast majority of miRNA expression profiles from solid tumor tissues and body fluids indicated that circulating miRNAs originate from tumor tissues and are protected from endogenous RNase activity reflecting the potential of developing circulating miRNAs as extracellular biomarkers of cancer and other diseases (Acunzo et al., 2015).

CRISPR Cas13a system

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CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems are originally derived from prokaryotic adaptive immune system against invading nucleic acid components. Generally, CRISPR/Cas system can be divided into two main classes, class I and II, according to the system comprising a single or multiple effectors (Liu et al., 2017). Among them, class II (e.g., Cas9, Cas12, and Cas13) possesses more widespread application, due to its simple components (a single effector protein and a programmable guide RNA, Wang et al., 2021).

Cas13 can be further divided into four subtypes, Cas13a-d, exhibiting diverse primary sequences except the two highly conserved HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domains, which are responsible for both cis- and trans-RNase activities (Florczuk et al., 2017). Structural studies revealed that Cas13a adopts a bilobed architecture including recognition (REC) and nuclease (NUC) lobes (Wang et al., 2021, Zhou et al., 2020).

CRISPR-Cas12,13,14 exhibits nonspecific degradation of non target (trans cleavage) after specific recognition of nucleic acids, thus CRISPR/Cas biology promises rapid, accurate, and portable diagnostic tools, the next-generation diagnostics. Cas13a due to its propensity to cleave RNAs after binding a user-defined RNA target sequence, is used to detect single molecules of RNA species with high specificity (Liu et al., 2017).

Microfluidics

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This multidisciplinary field refers to the science and technology of manipulating and allowing precise control of fluids in ultrasmall volumes (microliter, μL to femtoliter, fL), which are constrained in micrometer-scale channels (Zhang et al., 2019, Gyimah et al., 2021, Ronshin et al., 2022). Microfluidics deals with the management of liquids and particles on a scale of tens to hundreds of micrometers and of liquids and particles on a scale of tens to hundreds of micrometers and offers significant advantages over conventional macroscale systems (Gyimah et al., 2021), such as

An important aspect of microfluidic chip fabrication is the process that will be utilized. The possible solution for the device fabrication is the traditional method (Soft lithography) or Additive Manufacturing (FDM, DLP). The microfluidic device needs to fulfill certain parameters as Resolution , Surface Wettability, Transparency, Surface Roughness and Biocompatibility (Aladese et al., 2021).

Weaknesses in the RNA detection methods

The low abundance of miRNA in cytoplasm, extracellular vesicles (EVs), and body fluid usually presents challenges for its detection (de Planell-Saguer et al., 2013) Numerous nucleic acid amplification (NAA) methods and signal amplification approaches have been developed to improve the sensitivity and selectivity of miRNA detection, such as the quantitative reverse transcription-polymerase chain reaction (qRT-PCR), rolling circle amplification (RCA), exponential amplification (EXPAR), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP) (Shan et al., 2019).

These nucleic acid amplification (NAA)-based RNA detection methods are advantageous in terms of high fidelity, but they come across various disadvantages.

• intensive and time consuming
• highly sensitive to contamination with genomic DNA
• require the conversion of RNA to DNA and subsequent template replication steps
• sample loss issues
• amplification bias caused by error-prone sequence replication
• the highly homologous sequences of the miRNAs in one family

Our solution

Sometimes the solution of a problem can be achieved while thinking out of the Box!

This year our team decided to design an innovative diagnostic platform by replacing the widely used NAA-based RNA detection methods for signal amplification, with cutting edge approaches based on 3D-printed droplet microfluidics. Imitating the bio-inspired confinement effect, our diagnostic tool DIAS performs the CRISPR/Cas13a enzyme assay in picoliter-sized microreactors generated by droplet microfluidic chips. Specifically, the target miRNAs

together with the Cas13a mixture are mixed and emulsified with an oil generating multiple picolite-sized droplets. After recognition by the Cas13a/crRNA complex, the target miRNA induces the cleavage of the fluorescent RNA reporters that are located nearby the enzyme due to the trans-cleavage activity of the Cas13a. As a result, the fluorophore is released from the quencher thus getting rid of the fluorescence resonance energy transfer effect (FRET) and inducing fluorescence signals.

It is well known that the various interaction-based biochemical reactions occurring within the cell are very efficient, despite the minimal amount of substrate molecules participating in these reactions (Albayrak et al., 2016). This phenomenon is based on the confinement effect, which promotes the local molecule concentration for highly efficient reaction or detection.

As microRNA levels in plasma are extremely low, it is difficult to detect them in vitro without the aid of NAA. However, it is easy to visualize these microRNA molecules in a cell using fluorescent probe-based imaging techniques such as a fluorescence microscope (Baker et al., 2012). The accumulated fluorescent signal from a single RNA target-activated Cas13a's collateral cleavage is sufficient to illuminate a picoliter-sized droplet, thus enabling NAA-free and digital RNA quantification at the single-molecule level while succeeding excellent detection sensitivity and accuracy.

Specifically, the patient's blood samples are preprocessed with any commercially-available RNA isolation kit to extract the total RNAs. Then, the pretreated sample is mixed with the DIAS detection master mix which contains all the necessary reagents in specified preformulated proportions. Afterwards, the premixed test samples are inserted into a centrifugal microfluidic cartridge which is incorporated in a standard falcon tube. After processing at a fixed low centrifugal acceleration at a standard laboratory centrifuge, an emulsion is generated with fluorescent water-in-oil droplets. With one pipetting step the emulsion is inserted into a Neubauer improved counting chamber for fluorescent droplet read out using a simple fluorescent microscope. Based on the visualization-quantification software, the number of green fluorescent droplets is correlated with the amount of the target miRNA presented in the patient's blood which reveals the lung cancer probability score of the patient.

To sum up, DIAS is an in vitro diagnostic molecular device for the early detection of Non Small Cell Lung Cancer (NSCLC). Our CRISPR-based platform provides a costless, non-invasive, sensitive and point-of-care diagnostic solution for detecting abnormal serum miRNAs levels in patients with Non-Small-Cell-Lung Cancer during the first stages of the disease. On our journey of brainstorming, developing and experimentally testing our detection platform we had to follow a well-structure project pipeline based on the following successive steps:

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Our long term goal is the DIAS detection platform regulatory approval for NSCLC testing in diagnostic centers, labs and hospitals. The platform's unique advantages such as the low cost, the easy assay completion and high accuracy, could contribute to the incorporation of our test in the annual checkup examinations of people with high risk of lung cancer.

The lung cancer test points will receive our diagnostic tool as a formulated kit that will contain all system's components such as the reaction buffers, the microfluidic chip and the imaging software necessary for calculating the NSCLC diagnostic score after fluorescence microscopy analysis.

However, our vision is to develop a portable device for automated diagnostic score calculation based on the fluorescent droplet estimation and without the necessity of the fluorescence microscope. This portable device will use a small laser diode as a light source and a special detector camera for fluorescent droplets images acquisition and fluorescence estimation. In addition, different microRNA biomarkers could be detected by applying modifications to the crRNAs achieving multiplexing and expanding our system's application to different types of cancer or even to different diseases.

The future modifications to the existing structure of the DIAS detection platform will expand the system's implementation and applications constituting an all-in-one portable diagnostic system.