Background

Chronic hepatitis is a major global public health problem. Persistent inflammation can gradually damage the liver and lead to serious consequences such as cirrhosis and liver cancer. By 2040, deaths from chronic hepatitis will have exceeded those related to HIV infection, tuberculosis and malaria combined. In many cases, however, there are no obvious symptoms of liver dysfunction in the early stage of chronic hepatitis. For many patients, they don't even realize they need medical attention until it's too late.

So, we hope to offer a rapid home-based detection method for liver disease through synthetic biology, raising the public awareness of chronic liver disease.

Surveillance Indicators

Total bile acids (TBA) level, as a biomarker of liver function and hepatobiliary injury or disease, is an ideal preliminary diagnostic indicator of liver disease.

The current detection of bile acids mainly includes the determination of serum and urine. Serum bile acid content is inconvenient to sample and often affected by diet, so it is only suitable for accurate detection in hospital environment. By contrast, with the convenient sampling and stability against diet, urinary bile acid is more suitable for routine detection.

There are significant differences in urinary bile acid content between patients with liver disease and normal subjects. It can be seen from the table that TBA content (29.3 μmol/L) in the urine of patients with chronoc hepatitis is much higher than that of the people without cirrhosis (2.93 μmol/L), which is about 10 times. In addition, the content of each bile acid in the urine of patients with liver cirrhosis is also much higher than that of healthy people.

On this basis, we choose urinary bile acid as the detection indicator of chronic hepatitis. Furthermore, with the progress of the research, our project may develop into an early multi-disease diagnosis method.

Common detection methods

Researchers have been devoted to bile acid detection technology for decades, which can mainly be classified into chromatography and non-chromatography.

Chromatography methods are mainly used for laboratory research, of which Thin Layer Chromatography (TLC) and Electrospray Ionization (ESI)-MS are primarily applied to qualitative analysis. High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC)-MS and Liquid Chromatography-Mass Spectrometry LC-MS are used for both qualitative and quantitative analyses.

Non-chromatography methods are mainly utilized clinically, including the enzymatic method, Enzyme-Linked Immunosorbent Assay (ELISA) and Nuclear Magnetic Resonance (NMR). However, the concomitant high detection limit and low specificity are restricting their usage. In addition, these methods not only require specialized instruments and professionals to operate, but also cost considerable time and money, which makes it difficult for patients with chronic liver disease to be diagnosed at an early stage.

Therefore, our team tries to build a rapid home-based detection method for the preliminary diagnosis of some liver diseases and alert people to seek medical treatment in time, accordingly reducing the harm of liver diseases.

Solution

Since bile acids in urine are mainly sulfonated, we first use the sulfatase from Pseudomonas testosteroni to remove the their sulfate group. Secondly, it is reported that Farnesoid X Receptor (FXR) can dimerize Retinoid X Receptor (RXR) after being activted by the bile acids. So, we intend to use it as the detection device. Then, we link dimerization-dependent Red Fluorescent Protein (ddRFP) to FXR and RXR respectively (Design). They together construct a cell-free biosensor for the detection of urinary bile acid, achieving the goal of home-based preliminary diagnosis of liver diseases.

Our project tries to build a rapid and convenient preliminary diagnosis of chronic liver disease by means of synthetic biology, which is complementary to the existing clinical accurate diagnosis methods. We hope our project can provide a reliable solution to the World Health Organization's 2030 goal of eliminating hepatitis and reducing the incidence of liver disease.

Reference

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