(188cg) Ultrasensitive Microrna Detection for Disease Diagnosis

Ultrasensitive MicroRNA Detection for Disease Diagnosis

Currently,
disease diagnosis is facing two major problems: Inadequacy of early diagnosis
and high expenses. Lung cancer and colon cancer are two example cancer types which
suffer from the inability of current diagnostic methods (low-dose spiral chest
computed tomography and colonoscopy) for early detection, resulting in high
mortality rates. In addition, Alzheimer’s disease could benefit from early
diagnosis when some of the symptoms can be reversible, however, current
techniques (neurological exam, mental status tests, and brain imaging) are not
sensitive enough for early diagnosis. Mild traumatic brain injury is a
condition where the most accurate diagnosis methods are MRI (magnetic resonance
imaging) and CT (computed tomography), which are very expensive. Therefore,
there is a definite need for cheaper and more sensitive diagnostic techniques.

Recent developments in genome sequencing technology lead to the discovery of a new type of biomarkers, microRNAs
(miRNAs). miRNAs are small noncoding RNAs that have only 19-23 nucleotides. They have
an impact on many vital biological processes such as proliferation, metabolism,
immunological response, tumorigenesis, and viral infections with their ability to regulate gene expression. Also, miRNAs
are considered as biomarkers of diseases such as cancer, neurodegenerative
diseases (i.e. Alzheimer’s disease, traumatic brain injury, etc.)and cardiovascular diseases (i.e. heart attack),
tuberculosis and malaria sincethe levels of certain miRNAs change
according to presence and stage of the diseases. Moreover, they are present and
stable in many body matrices such as
tears, saliva, sputum, breast milk, urine
and seminal fluid, which makes them
advantageous biomarkers that can be screened from multiple places.

The
important task of specific and sensitive miRNA detection is hindered by
challenges in consistent and accurate miRNA amplification. MicroRNAs are small
and similar in sequence with as little as one nucleotide mismatch between
different miRNAs, such that it is difficult to detect them with high
specificity. This is especially difficult considering that many miRNAs are
present in very low concentrations, especially in the early stages of a
disease. Currently, the gold standard for miRNA detection is RT-qPCR (reverse
transcriptase quantitative polymerase chain reaction). RT-qPCR can successfully
detect miRNAs with high sensitivity and specificity. However, it is a
time-consuming (6-7 hours) and expensive method that is not applicable for low
resource settings since it requires a thermocycler, a trained technician and, a
laboratory.

In this study, we developed and characterized a new
tunable DNA amplification chemistry, UDAR (Ultrasensitive DNA Amplification
Reaction), that mimics the switch-like characteristics of biological systems
such as cell signaling and genetic regulation. UDAR is an isothermal reaction,
which eliminates the need for a thermocycler. This high-yield switch creates a
definitive change in fluorescence output and can be imaged using a cell phone. Currently, 3 different miRNAs were
successfully detected with UDAR, with a limit of detection on the order of 10 fM. In the future, UDAR can be converted into a quantitative assay by counting single
amplified miRNA molecules with the help of digital microfluidic tools. The
aim of this study is to develop a novel miRNA detection method that is simple,
inexpensive and rapid without compromising from sensitivity and specificity.  Our ultimate goal is
to develop a miRNA assay that can be used in doctor’s office or in limited
resource settings.