Background


Major depressive disorder (MDD), or clinical depression, is a complex and heterogeneous clinical syndrome characterised by persistently depressed moods, loss of interest in daily activities and physical symptoms such as appetite changes, sleep pattern changes, and fatigue according to the widely used DSM-5-TR criteria[1]. It currently affects at least 280 million people worldwide [2], and according to the World Health Organisation (WHO), it is predicted to be the leading cause of disease burden worldwide by 2030 [3].

Figure 1 : Increase in mental health morbidity in Kerala
Clinical depression is a very prominent disease. In our state, Kerala, alone, the prevalence of mental disorders was reported to be 12.43% in 2017. The number of individuals with mental health illnesses in the state increased from 272 per 100,000 to 400 people per 100,000 from 2002 to 2018 [4]. The COVID-19 pandemic has profoundly impacted mental health in Kerala and globally. Isolation, grief, economic challenges, and uncertainties related to the pandemic have increased the risk of depression and exacerbated existing mental health conditions. A recent study found that during the COVID-19 pandemic in Kerala, depression (75.2%) and anxiety (69.4%) were significant problems faced by people under home quarantine [5]. The state experienced a rapid rise in mental health morbidity in recent years.

Mental health and psychological care support was revealed to be vitiated by a scarcity of resources and inadequate information. Following the pandemic, the global prevalence of anxiety and depression increased by a massive 25%, according to a scientific brief released by the WHO [6]. Millions of people with depression suffer because they do not have access to professional help that starts with a diagnosis, a trend observed in our locality as well [7]. For a disorder that is so common, even in people around us, it is heavily misunderstood.

Depression is as much a global issue as it is a local issue. One that is becoming increasingly important to address.


Inspiration


Early in the ideation process, our team ran into multiple news stories about the increasing suicide rates in higher educational institutions across India [8] [9]. We learnt how depression is one of the leading causes of suicide in India and across the world [10], especially in recent years. The growing concern about mental health issues amongst students strengthened our desire to work on clinical depression.

After a remembrance ceremony conducted by the Student Welfare Council of IISER TVM following the loss of a student to suicide, one of our team members opened up about their struggle with depression. Their experience opened our eyes to the silent battle fought by people suffering from MDD—stories that often go unheard. They recounted their multiple inconclusive doctor visits and difficulty receiving a proper diagnosis. Learning about their experiences helped us understand the process of diagnosing depression and its shortcomings better.

We realised the need for an objective tool considering the biological aspects of clinical depression. As we started our work, we looked to other iGEM teams for inspiration. We found that Team Moscow 2021 (miPression) worked extensively on a diagnostic kit to distinguish depression from other mental disorders. Their project validated our initial ideas for a diagnostic kit and prompted us to dig deeper into the problem, helping shape our project design [11]


The start of OASYS


We consulted three professional psychiatrists from the Government Medical College (GMC), Thiruvananthapuram, Kerala, India, to study the scale of the issue. We learnt about the current methods of diagnosis of MDD, which mainly consist of self-report questionnaires, which consist of varied questions designed to screen the patient for MDD. The questionnaire is usually followed by clinical interviews with a Mental Health Professional (MHP), who often relies on symptoms reported by the patient or the people around them. Non-verbal cues such as body language and voice modulation, along with the questionnaire results, are assessed, and the diagnosis for MDD is made based on the DSM-5-TR criteria [12]

Psychiatrists from GMC told us about how often they meet patients who confess to trying to manipulate questionnaires for fear of being diagnosed with depression. They also spoke about the stigma associated with mental illnesses, particularly depression. We learned that the current methods could be subjective and prone to variability, and this could lead to misdiagnosis. Patients and the people around them are often ignorant of depression and other mental illnesses as diseases and are hesitant to accept their diagnosis.

We realized there is a need for an objective and sensitive point-of-care diagnostic aid for clinical depression. And that’s what we set out to build - OASYS. Through OASYS, we hope to offer an objective aid to simplify the process of MDD diagnosis by reducing the dependence on self-reported symptoms and making it less time and effort-intensive for the patients [13].


So, what is OASYS?


OASYS is a novel and objective FRET (Förster or fluorescence resonance energy transfer) -based microfluidic device that can quantify and analyze biomarkers such as neurotransmitters, microRNAs, hormones, and proteins in human peripheral blood serum. We use this device as an objective aid to diagnose Major Depressive Disorder (MDD).

We have chosen five putative blood biomarkers correlated to MDD - the neurotransmitter serotonin, the hormone cortisol, a novel protein - Gs alpha, and two microRNAs - 124 & 132, which were chosen together through a Bayesian analysis . We have developed two quantification methods - Aptasensors for serotonin, cortisol and the Gs alpha protein, and Magnetic Nanoprobes, used for the microRNAs, that work on the principles of FRET, producing fluorescence whose readouts can relate to the blood biomarker levels.

These quantification systems are incorporated into a microfluidic chip that can take blood samples as input and run the assays simultaneously. We built a hardware system with fluorometric sensors that can detect the fluorescence produced by the chip to analyse and estimate the biomarker concentrations.

OASYS is more than just a diagnostic aid; it can also be a powerful research tool. Tailored to be specific to any biomarker, our tool simplifies data collection of biomarkers from blood samples for population studies. It can also be used to monitor the efficacy of antidepressant treatment in patients by analysing fluctuating blood-biomarker levels. It could assist clinicians in personalising the drug combinations and dosage, catering to the individual's needs and paving the way for personalized medicine.


Is this actually possible?


Diagnosing a disorder such as clinical depression is highly challenging for multiple reasons. It is a very heterogeneous disease, showing up differently in different people. The causes for MDD can vary immensely, such as anxiety/trauma-induced depression, genetic disposition, and other biological factors such as vitamin D deficiency and bacterial infections. Some objective tests, such as the complete blood account with a comprehensive metabolic panel, are done to rule out any organic or medical causes of MDD [12].

For OASYS to function as a diagnostic tool for MDD, we need to know the exact "cut-off" or threshold values for each biomarker level beyond which (either above or below) the patient can be considered at high risk for MDD. This requires collecting and analysing population data to find a suitable threshold.

Talking to multiple psychologists, psychiatrists, researchers and mental health professionals, we realised that currently, there are various problems with the reliability of the biomarkers:

  1. The exact effect each biomarker has on MDD is unknown. Biomarkers are not causative, only correlated to MDD. Building the relationship between absolute biomarker levels and the severity of a mood and symptom-based disorder like clinical depression is complex [14].
  2. There is a severe lack of data on biomarkers related to mental illnesses, especially for MDD [15].
  3. We learnt that so far, all studies have been based on clinical trials with small sample sizes. Additionally, their relevance is restricted only to a small population; hence, we cannot make generalised conclusions about how our chosen biomarkers co-relate to MDD.
  4. Progress on biomarker research for MDD has been slow, and as it stands today, biomarkers available in the literature aren't reliable enough to be used for diagnosing depression yet [15].

As Dr. Varsha Singh, a cognitive psychologist from IIT Delhi, says, "Biomarker research on mental illnesses needs to catch up with research on other diseases."

Considering the inputs given to us by professionals from various fields and serving as an essential milestone in our project journey, we realised that our project should change from focusing on biomarkers to building technology that can analyse fluctuating biomarker levels and be put to varied applications.


OASYS can help!


Despite the prevalence of mental illness, progress in developing accurate and objective diagnostics has been lagging. Some laboratory tests, such as the dexamethasone suppression test (DST), are available to aid in diagnosing depression; however, they are not viable due to high costs or slow results [16].

We realised that single biomarker tests can’t accurately diagnose a disorder as heterogeneous as MDD and that there is a need for a cheap, user-friendly and accessible quantitative test.

OASYS is a novel microfluidic device and sensor-based tool that simultaneously detects various biomarkers through two quantification systems and produces fluorescent readouts that can further be analysed for biomarker concentrations. To seamlessly integrate this tool into real-world clinical settings, complementing currently available diagnosis methods, OASYS was designed to be compact, modular, cheap, accessible and easy to use.

It can be used for various applications in different settings and even as a research tool to help it become a diagnostic aid.

Find out more about the uses of OASYS on our Implementation page!


OASYS Components


The Biomarkers

Neurotransmitter - Serotonin

Neurotransmitters are endogenous chemical messengers that help neurons throughout the body communicate with each other. Neurotransmitters amplify, transmit, and convert signals in cells, and through chemical synaptic transmission, they help with various brain functions.[19] We use the neurotransmitter serotonin (5-hydroxytryptamine) as the first biomarker in our kit. Serotonin levels in the blood have been shown to decrease in patients with depression. [18]

Hormone - Cortisol

The second biomarker is cortisol, widely known as the body's stress hormone. Hormones are chemical messengers found in multicellular organisms that affect psychology and behaviour. Cortisol is a steroid hormone in the glucocorticoid class that plays an important role in stress response. Studies indicate that higher cortisol levels in blood are well correlated with the occurrence and severity of MDD. [19] [20]

Protein - Gsα subunit

The GSα protein is our third biomarker. G proteins, also known as guanine nucleotide-binding proteins, are proteins that serve as a molecular switch inside cells, transmitting signals from external stimuli to the cell's interior. G-proteins and RGS (Regulators of G protein signalling) proteins are two protein families that are deeply engaged in both the onset of depressed states and the action of antidepressant medications. This novel biomarker was shown to be present in lower concentrations in clinically depressed patients. [21]

miRNAs -124 & 132

MicroRNAs (miRNAs) are small non-coding RNAs. Most miRNAs are transcribed from the DNA sequence into primary miRNAs and then processed into precursor and mature miRNAs. Our project makes use of miRNAs 124 and 132. Multiple studies have observed the upregulation of these miRNAs in clinically depressed patients.[22][23] We chose miRNAs as they are well-studied and easily accessible from blood, serum and other body fluids.


The Quantifiers


Aptasensors

Figure 4 : Magnetic Nanoprobes - Tian, H., Yuan, C., Liu, Y. et al. A novel quantification platform for point-of-care testing of circulating MicroRNAs based on allosteric spherical nanoprobe. J Nanobiotechnol 18, 158 (2020). https://doi.org/10.1186/s12951-020-00717-z

Aptamers are artificially synthesised single-stranded DNA or RNA sequences that can easily change conformations, allowing them to bind to specific targets with high affinity. Hence, they can be used in biosensing for clinical diagnostics. FRET is a non-radiative energy transfer process that occurs over small-scale separations (usually in nanometers) between an emitter (donor) and an absorber molecule (acceptor), often called a ‘FRET’ pair. We have chosen a fluorophore and a quencher as the donor-acceptor FRET pair.

For our project, we make the use of Aptasensors or FRET aptamers. The Aptasensor comprises of an aptamer, which contains a fluorophore, and the cDNA, which has a quencher attached to it. While the cDNA is bound to the aptamer, the fluorophores transfer energy to the quencher, and fluorescence is not observed. However, in the presence of the target, the FRET aptamers bind to it, displacing the cDNA. With no quencher in the vicinity, the fluorophore will produce a detectable fluorescent signal. These FRET aptamers are used to quantify the neurotransmitter serotonin, the hormone cortisol, and the GSα protein[24].

Magnetic Nanoprobes

Nanoprobes are small, nano-sized devices that interact with a biomolecule and can be used for diagnostic purposes. Our nanoprobe comprises a magnetic nanoparticle with attached molecular beacons that work on the principle of FRET and produce fluorescence through a fluorophore-quencher pair. Our molecular beacons are single-stranded DNA structures with three prominent parts: one complementary to the target miRNA, one attached to a fluorophore, and one attached to a quencher.

When inert, the nanoprobe exists in a hairpin structure, and the probe holds the quencher near the fluorophore, thus emitting no signal. When the target miRNA binds to the probe, the target-probe hybridisation can open the hairpin, forming an active "Y" structure. This active structure separates the fluorophore and the quencher, emitting fluorescence. We will further enhance the detection of the miRNAs present in lower concentrations in the blood by attaching the molecular beacons to magnetic nanoparticles to focus fluorescence on a smaller area [25].

How OASYS Works


Microfluidic Chip

We are designing a microfluidic chip that uses human blood samples as the input and incorporates all the quantification systems for the biomarkers. It is made of PolyDiMethylSiloxane (PDMS), a silicone polymer. [32]


Figure 6 : Original microfluidics chip design using 3dµ f software by CIDAR Labs


The design involves five channels, one for each biomarker. The blood sample is introduced to the main port, which is equally divided into all the channels through valves and pumps. Each channel has different chambers, called mixers, that contain the quantifiers: magnetic nanoprobes for miRNAs and FRET aptamers for serotonin, cortisol, and Gsα. These mixers are chosen based on the specific conditions required for the quantifier to attach to the biomarkers. Once the quantifiers bind to the biomarkers, we flow them into transparent chambers where fluorimetric sensors can sense the fluorescence and give us a measurement of the biomarkers.[33] Read more about it on our Microfluidics Page.

Detection System

OASYS comes with a fluorescence detection system along with the microfluidic chip. The detection system consists of a 3D-printed box equipped with an LCD screen, an Arduino UNO setup, and the necessary accessories.

When the microfluidic chip is inserted into the device, the internal blue LED excites each of the five samples, and the fluorescence emitted is detected by a different photodarlington sensor. The intensity of fluorescence measured is then converted into the biomarker concentration levels and displayed on the LCD screen, with all data readings being managed by the Arduino UNO microcontroller. Learn more about how the detection system works here!


Conclusion


Our project, OASYS, consists of a microfluidic device with two different fluorescence-based quantification systems to measure the levels of five blood biomarkers correlated to MDD. It comes with a hardware system that can detect the fluorescent readouts and relate them to the biomarker levels in the blood. OASYS is built to be used as a diagnostic aid for clinical depression, with its applications extending to prognosis, antidepressant tracking, clinical drug trials and advancing scientific research. Through our project, we recognise the importance of science and synthetic biology communication and the need for mental health awareness.


References


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