Principle

Our project designed a microbial sensor based on a nucleic acid aptamer and modified the bacteria on the complementary chain of the nucleic acid aptamer. After the aptamer binds to the complementary chain, the complementary chain is competed with the aptamer by the high affinity of the target protein and the aptamer, We culture the released complementary streptavidin complex in vitro, and use the bacteria's own proliferation to amplify and convert into fluorescent signals. The design of microbial sensors is divided into two key components: aptamers and microbial fluorescent probes.

Aptamer

Aptamers are a class of single-chained oligonucleotides (DNA, RNA, modified RNA) that bind specifically to a variety of target molecules with high affinity. Due to the excellent characteristics of the aptamer, almost all aptamers can be used in applications where antibodies are involved.

Aptamers some advantages over general antibodies and enzymes:

The binding of aptamer to various target molecules is based on the diversity of single-chained nucleic acid structures and spatial conformations. It can be adaptively folded to form some stable three-dimensional structures binding to their target molecules by the pairing between certain complementary bases in the chain as well as electrostatic interaction, hydrogen bonding, etc, therefore showing a very high affinity for their target molecules, and this high affinity is usually higher than the complementary chains that are complementary to the base of the aptamer, thus causing a break in the hydrogen bond between the aptamer and the complementary chain.

We select CK-MB protein, a bio-marker for the diagnosis of acute myocardial infarction as a test substance to test our microbial sensors. We screen its aptamer and design its complementary chain (CS-15) to build our microbial sensor.

CK-MB protein is an isoenzyme of creatine kinase (CK), also known as creatine phosphokinase (CPK), with a molecular weight of 81000, consisting of two subunits. There are four isoenzyme forms: muscle type (CK-MM), brain type (CK-BB), hybrid type (CK-MB) and mitochondrial type (CK-MINI).Creatine Kinase-MB (CK-MB) is a biomarker used to diagnose acute myocardial infarction with a molecular weight of 44 kDa.Clinically, the reference range of creatine kinase isozyme was 2.0-25.0 U/L when measured by enzyme activity method, and the upper limit of normal value was 20 ng/mL when measured by mass method.

Microbial Fluorescent Probe

Fluorescent probe method is the qualitative or quantitative analysis using the characteristic fluorescence of the fluorescent substance under the external energy acts. The microbial fluorescent probe utilizes the self-proliferation of the engineered bacteria to further increase the amount of fluorescence, thereby providing a more favorable environment for the subsequent detection.
The microbial fluorescent probe itself has a short manufacturing time and is relatively stable and has more ideal robustness. Besides, we take advantage of the convenient detection of microbial probe to bio-magnify through microorganisms (E. coli) to facilitate the detection while this process can just be achieved by microbial amplification. In addition, by constructing recombinant plasmids, we were able to expand the signal a step further. It is worth mentioning that microbial fluorescent probes can construct gene lines expressing fluorescent proteins of different colors, thus achieving joint detection of markers of different diseases or different markers of the same disease.

We construct three gene lines to express green, red, and yellow fluorescence respectively to enable quantitative detection of three proteins.

First, we use SMPB (a sulfhydryl-amino crosslinker) to link the engineered bacteria to the complementary chain (CS-15) with a thiol group modified at the 5' end. And then we use magnetic beads modified with streptavidin to bind biotin-modified aptamer. Finally, the two parts react at room temperature to get our microbial sensor.

During the detection process, we added a sample to the microbial sensor solution, in which CK-MB protein was combined with the aptamer, so that CS-15 was competitively shed. The more CK-MB, the more CS-15 was competing, The more the number of engineering bacteria.
By magnetic adsorption, the reaction system will be divided into two phases: one is the solid phase mainly composed of magnetic beads and aptamers, and the other is the liquid phase with engineering bacteria. The liquid phase is cultured in the medium. Due to the difference in the number of engineered bacteria under the competition of CK-MB protein, after the same time of culture, the optical density and fluorescence intensity will be different, so that we can quantitatively detect CK-MB protein.

Reference:
[1]Darwish N T, Sekaran S D, Alias Y, et al. Immunofluorescence-based biosensor for the determination of dengue virus NS1 in clinical samples.[J]. Journal of Pharmaceutical & Biomedical Analysis, 2018, 149:591.
[2] Hafner G, Peetz D, Dati F, et al. Analytical and clinical evaluation of troponin I determination on dimension RXL-HM.[J]. Clinical Chemistry & Laboratory Medicine, 2000, 38(4):355-361.
[3] Tiwari A, Dhoble S J. Recent advances and developments on integrating nanotechnology with chemiluminescence assays[J]. Talanta, 2018, 180:1.
[4] Perret G, Boschetti E. Aptamer affinity ligands in protein chromatography.[J]. Biochimie, 2017, 145.
[5] Jayasena S D. Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics[J]. Clinical Chemistry, 1999, 45(9):1628.
[6] Citartan M, Gopinath S C, Tominaga J, et al. Assays for aptamer-based platforms[J]. Biosensors & Bioelectronics, 2012, 34(1):1-11.
[7] Poiata E, Meyer M M, Ames T D, et al. A variant riboswitch aptamer class for S-adenosylmethionine common in marine bacteria.[J]. Rna-a Publication of the Rna Society, 2009, 15(11):2046-56.
[8] Jo H, Her J, Lee H, et al. Highly sensitive amperometric detection of cardiac troponin I using sandwich aptamers and screen-printed carbon electrodes.[J]. Talanta, 2017, 165:442-448.