Team:ZJU-China/Safety
Our project aims to detect the injury based on a cell-free device with three connected enzymes which fixed on a biofilm scaffold. We use blood specimens with biochemical analysis to calibrate our device and help us with our machine learning modeling to set a diagnosis criterion.
Our biofilm scaffold consists of curli which is the protein found on the surface of E.Coli. Curli may facilitate bacteria’s invasion into host cells and activate corresponding cytokines and inflammatory mediators in plasma. But the system we wish to produce will be ultimately cell-free and no living organisms will be included. Besides, curli will be immobilized and covered by Nafion on the electrode, so contact with curli will not happen. Therefore, biofilm scaffold is relatively safe.
Blood SamplesWe have gotten approval from our university authority and collected some blood samples from SIR RUN RUN SHAW HOSPITAL, which is classified as a Grade 3 Class A hospital by Chinese government. The procedure of blood sampling was done by medical personnel using ideal blood collection equipment. Besides, our experiment was conducted under professional doctors' supervision and waste was collected by the specialties in the hospital. (It is also an essential part of Integrated Human Practice.)
Our project meets ethical requirements. Blood samples we got from hospital are all remaining samples after satisfying patients’ pathological diagnosis need. We keep secret of patients’ privacy information. We conduct experiments with clinical material following our country's laws and our university's rules.
The main issue for work with blood samples is the potential for infection. Clinical samples may be contaminated with pathogens. The high risk, well known viral agents such as Human Immunodeficiency Virus (HIV), Hepatitis B virus (HBV) and Hepatitis C Virus (HCV) are not the only agents that may be present in blood. Other viruses such as HTLV1 and B19 as well as various bacterial agents may also be present. Regarding the likely incidence of a pathogen in blood samples, several factors should be considered. These include known medical history of a patient or donor, whether the samples are from individuals showing clinical symptoms of infectious disease, the incidence of the various pathogens that are endemic in the local population or donor group and the type of sample. Fortunately, SIR RUN RUN SHAW HOSPITAL offered us the physiological indices of blood samples and other relevant information. So, the samples we used are from formal approach and relatively less dangerous. To minimize the risks，we have made Standard Operating Procedure (SOP) which is strictly followed in our iGEM lab.
Standard Operating Procedure (SOP)As insurance, Blood samples from all patients should be considered infective. The following precautions followed in our lab are also recommended for all health-care workers in clinical laboratories.
1. All the experiments where blood is involved must be conducted in a separate room within the laboratory.
2. All specimens of blood should be put in a well- constructed container with a secure lid to prevent leaking. Care should be taken when dealing with each specimen to avoid contaminating the outside of the container and of the laboratory form accompanying the specimen.
3. Lab surface including correlated equipment is decontaminated with chemical disinfectant prior and after disposal.
4. Every piece of material that has been in direct contact with blood should be disposed of correctly via the clinical waste route when work activities are completed.
5. All persons processing blood specimens should wear gloves. Gloves should be changed, and hands washed after completion of specimen processing.
6. Use of sharp objects (e.g. needles, syringes, scissors) should be limited for fear of injuries and cross infection.
7. All persons should wash their hands after completing blood specimens’ processing.
Measure the plate in plate reader, the excitation filter was set to 485nm/10nm and the emission filter was set to 525nm/10nm. Pathlength correction was turned off. The gain setting was 50. Fluorescence was from the top. The temperature setting was 26.6°C. Record the data.
• Fluorescence standard curve
Spin down fluorescein stock tube. Prepare 10x fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1xPBS. Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 μM.
Prepare the serial dilutions of fluorescein as shown below. Set 4 copies.
Fig.2 Dilution of fluorescein [1]
Measure the plate in plate reader, the excitation filter was set to 485nm/10nm and the emission filter was set to 525nm/10nm. Pathlength correction was turned off. The gain setting was 50. Fluorescence was from the top. The temperature setting was 26.6