PROJECT DESCRIPTION

The problem

Currently, sepsis diagnosis depends on clinical judgment and standard lab tests, which can take up to 72 hours to bring results. However, even a one-hour delay in diagnosis can increase the risk of death by 6–10%. (1)

Current laboratory techniques for sepsis diagnosis

Blood culture

Traditional blood cultures remain the gold standard for pathogen identification, but they can take more than 72 hours to yield results. (1)

PCR

PCR-based microbial identification when combined with mass spectrometry, significantly reduces turnaround time to 6–8 hours. While faster than blood cultures, this timeframe still falls short of the 1–3 hour window that clinicians aim for to guide early sepsis management. (1)

Current biomarkers for sepsis diagnosis

C-reactive protein (CRP) increases slowly after infection, often peaking up to 72 hours, and lacks specificity for sepsis. CRPs levels also poorly correlate with disease severity. Procalcitonin (PCT), rises faster than CRP, typically peaking around 24 hours after bacterial infection, but this delay may still hinder timely diagnosis if relied upon alone. Both conventional biomarkers have moderate specificity and sensitivity for sepsis diagnosis and prognosis. (1)

"The development of sensor technologies which evaluate levels of sepsis biomarkers with fast analysis times has the potential to revolutionise sepsis diagnosis and management." (2)

AECHMI means in greek the point of a sharp object. It symbolizes the sharpness in precision of our diagnostic approach and was also chosen to describe the sharp microneedles of the device our system will be incorporated in.

AECHMI tackles the main problems in sepsis diagnosis:

Aechmi, combines the specific recognition of microRNA sepsis biomarkers using the CRISPR/Cas13a system, with an isothermal signal amplification method, enabling the detection of low biomarkers concentrations in real time.

Τhe electrochemical signal produced will be processed and interpreted by a software, leading to a diagnostic conclusion, insights into disease severity and early targeted treatment. READ MORE

In the next stage of development, our biological system is going to be incorporated into a microfluidic-microneedle portable device, enabling the rapid, real time detection of biomarkers inside and outside hospital settings. READ MORE

Circulating microRNAs

"miRNAs have been described in the blood of patients with inflammatory/infectious diseases, suggesting that circulating miRNAs might also be suitable as biomarkers in the setting of critical illness and sepsis."

MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression. Specifically, they bind to the 3′ untranslated region (3′UTR) of target mRNAs, inhibiting their translation. A single miRNA can influence the expression of hundreds of mRNAs, placing miRNAs at the center of complex regulatory networks that control both physiological and pathophysiological processes. Aberrant miRNA expression has been reported in tightly regulated conditions such as sepsis. Importantly, miRNAs can be detected in the bloodstream, where they remain remarkably stable under conditions that would normally degrade most RNAs. This stability is conferred by their association with RNA-binding proteins, lipoprotein complexes, or encapsulation within microparticles. Motivated by this, we analyzed datasets and reviewed the literature to identify circulating miRNAs in blood with dysregulated levels during the early stages of sepsis. (7)

CRISPR/Cas13a system

"The CRISPR/Cas13a system has been developed as an effective tool for molecular diagnostics, with expectations to improve the sensitivity, specificity, operability, portability and cost performance of molecular diagnostics tools."

Originating from the natural adaptive immune system of prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas endonucleases protect cells against invading genetic elements. While some Cas proteins act on DNA, Cas13a is an RNA-guided ribonuclease that can be programmed with CRISPR RNAs (crRNAs) to selectively recognize RNA targets. (3) This unique property has made Cas13a particularly suitable for the detection of microRNAs. Cas13a possesses has the ability for cis-cleavage of the target RNA and trans-cleavage of non-specific RNAs.Through cis-cleavage the specific recognition of target microRNAs is achieved,while through trans-cleavage of non-target RNAs the signal amplification is achieved, increasing the sensitivity on disease diagnosis. (4)

Catalytic Hairpin Assembly

"The Catalytic Hairpin Assembly (CHA) signal amplification strategy has shown great potential in enhancing sensitivity, as it is an efficient, enzyme-free, and isothermal amplification technique. Using CHA, a hundred-fold signal amplification can be achieved, and it has been widely applied in the construction of sensing systems for miRNA detection." (8)

Our biological system is designed for integration into a microneedle–microfluidic device. A major challenge in such devices is insufficient sensitivity, as microRNAs are often present at low concentrations in biological fluids. (8) To address this limitation, we employed catalytic hairpin assembly (CHA), an enzyme-free, isothermal signal amplification method based on two partially complementary DNA hairpins (H1 and H2) and a single-stranded initiator oligonucleotide. Incorporating Catalytic Hairpin Assembly enhanced the sensitivity of CRISPR/Cas13a collateral cleavage activity, enabling earlier and more reliable biomarker detection. (5)

References