Product DesignSynthetic biology products have limited reaction platform because of the biosafety issue of using living cells, and the difficulties to store in vitro liquid reaction system. However, a well designed paper chip can break the limits. The DNA device and proteins that required for detection can mix with E.coli cell-free system, freeze-dried on a paper chip, and stored in low temperature. The blood sample from the patient provides a liquid environment which can reactivate the cell-free system. The reaction takes place when the target sequence presents, emitting fluorescence on screening chip, which can yet be regarded as a good product direction.On the other hand, the project is intended to simplify the process of detection of lung cancer. Therefore developing a paper screening chip can provide effective detection as well as simplifying the processes of product making, transportation, and preservation.
Cell-free systemA cell-free system is an in vitro tool widely used to study biological reactions that happen within cells while reducing the complex interactions found in a whole cell. However, some of its characteristics made it become a popular environment when some real product is going to develop and put on the market. In 2016, Keith Pardee and his team develop a paper-based biomolecular sensor for zika virus detection. In the paper, they used the cell-free system as an environment and put enzyme, sensor, extra material within it. With the support of NASBA technic, they can amplify the molecule and detect zika virus directly with patient’s blood serum.
NASBANucleic acid sequence based amplification (NASBA) is a method in molecular biology which is used to amplify RNA sequences under a constant temperature. This process starts with reverse transcription of an RNA sequence using a designed reverse primer to create duplicated RNA or DNA sequences. Then RNase will degrade the RNA sequence. After that, a primer which contains T7 Polymerase will bind to the section and will elongate the sequence into complementary strand. Compared to Polymerase chain reaction(PCR), this method does not require a PCR machine, which can be innovated easily and may be designed into an easy kit for daily detection in the future.
-Extract few drops of patient’s blood
-Centrifuge blood to separate the serum layer by using a hand-power centrifuge
-Extract serum layer and add it in to the NASBA reaction tube
-After finishing the NASBA process, add few drops of NASBA product onto the paper chip
-Wait for the testing result. Fluorescence or color indicates the present of EML4-ALK mutation.
For our project, we need to have an environment which both transcription and translation process can be started automatically. As thinking about biosafety, we decide to adopt a cell-free system to prevent the patient from interacting with virus cell directly. For using the cell-free system effectively, we decided to make the product into test papers. We chose test papers because they can be massively produced and are easy to store. So it will be cheap to make and to buy in the future. Also, our test paper can change target easily which will be suitable for detecting different kinds of DNA in blood. (See the result session for demonstration)