This year, ShanghaiTech_China designed a hero fighting against the devil of antibiotic resistance, whose name is CESAR.
CESAR, Cas12a-based Effective Solution to Antibiotic Resistance.
With designing CESAR, we first introduced Cas12a system into the field of antibiotics-related combined detection.
CESAR-I & CESAR-II
CESAR contains two devices with different purposes:
*CESAR-I is a portable and rapid-response device for on-site antibiotic-residue detection. By integrating the aptamer-sensing module, Cas12a-reporting module, and fluorescence-measuring module, it simplifies the process and lowers the cost of antibiotic-abuse surveillance.
*CESAR-II is designed for doctors' detecting ARGs. It has RPA amplification module, Cas12a-reporting module, and fluorescence-measuring module. It is cost-effective and can help eliminate the medical disparity caused by unbalanced economic development.
Figure 1. The schematic flow of CESAR.
Four modules of CESAR
There are 4 modules we designed for the two devices. Among them, 2 modules are shared by CEASR-I and CESAR-II. This design can increase the inclusiveness of our device: by changing one or two modules, CESAR can do much more detection tasks than our original expectation.
Once users added samples into CESAR, the whole process can be divided into three steps: pre-detection step, detection step and analyzing step.
In order to further understand the problem, based on our understanding, we feel that the widespread of antibiotics might be from the animal husbandry industry, and its primary victim will be our medical system. Therefore, we consulted professions for a thorough analysis of our issue.
In the pre-detection step, the samples will be prepared for Cas12a detection.
This module is designed for CESAR-I. The reaction materials contain aptamer for some specific antibiotic molecules. The aptamers are pairing with activator DNA, forming a sandwich structure (refer to Engineering Success for details), before adding samples. If the added samples contain antibiotic residues, the aptamer will bind them, releasing activators. The more antibiotics it contains, the more activator will be released. Now antibiotic molecule signal has been transformed to nucleic acid signal, which will then be detected by Cas12a.
Figure 2. The Aptamer Sandwich.
This module is designed for CESRA-II. The reaction materials contain RPA enzymes and buffer. Because CESAR-II detects antibiotic resistance genes, which are nucleic acid signal themselves, this module does not need signal transformation. However, antibiotic resistance genes in bacteria are not as large as activators in amount. So rather than signal transformation, we need a signal amplification in this module. After RPA amplification, the antibiotic resistance genes will then be detectable for Cas12a.
In the detection step, the nucleic signals will be detected by Cas12a.
In this module, Cas12a will recognize activator DNAs or antibiotic resistance genes, which are products of the former step. These products will activate Cas12a, and then Cas12a will have the trans-cleavage activity (refer to Proof of Concept for Cas12a introduction). With this activity, Cas12a can cut non-specific single-strand DNA (ssDNA), which is artificially synthesized and modified by fluorescence groups. The ssDNA reporter has a fluorescence group (FAM) at one end and a quenching group at another end (BHQ1). It will emit fluorescence after being cleaved. By now, the signal has been transformed to fluorescence signal, which can then be detected by electro-fluorescence reading elements.
In the analyzing step, the fluorescent signal will be read analyzed.
In this module, the fluorescence will be read and analyzed. According to users’ actual needs, we provide two options: one with only a line of small windows for users to read the signal by naked eyes; the other one with an extra micro-chip that can electrically measure and analyze the fluorescence intensity. The two options are designed for qualitative or quantitative detection, respectively.
Figure 3. The fluorescence result we got, signal intensity changes along with sample concentrations.
The design of the whole device
The whole device is a small box, with an electromagnet on the lid, a rack for placing combined tube and a space for exciting light and analyzing chip. The other two elements: several small rings holding reaction materials and the combined tubes are disposable. The reaction materials for the first reaction step, the pre-detection step, have been already fixed in the combined tubes, which contain RPA systems with buffer or aptamer system with activator. The reaction materials for the second reaction step, the detection step, are fixed on the special designed rings. The rings have iron in it, so will be held by the electromagnet. When the first step reaction finished, the electromagnet will power off and the rings will fall into the tubes. Then the reaction materials will be mixed with products of the first reaction. Now if the target is in the sample, the Cas12a will be activated and cleave the ssDNA reporter. Which will then be measured and analyzed by the fluorescence-measuring module. Users can also observe the fluorescence through naked eyes.
Figure 4. The outlook of CESAR.