1. Background
At present, the diagnosis of major depression disorder (MDD) is mainly based on clinical symptoms, supplemented by self-assessment scale, and lacks objective bio-molecular markers, resulting in misdiagnoses, and mild gradually into severe illness, even leading to suicide.
Our project is to develop a diagnosis platform using microRNA (miRNA) as bio-molecular marker for MDD diagnosis, since the objective diagnosis of MDD is very important before treatment.
Please see our project description.
2. Develop a paper strip sensor based on cell free system to detect miRNA through the expression of colored protein for MDD diagnosis
Construction and identification of toehold switch plasmids
We designed 3 toehold switch plasmids with target sequences of miR-34a-5p, miR-221-3p and let-7d-3p. These toehold switch plasmids were identified by restriction endonucleases (EcoR I and Hind III) digestion assay (Fig.1).
Fig.1 Identification of pET-28a-toehold switch-amilCP, pET-28a-toehold switch-mRFP and pET-28a-toehold switch-amilGFP.
M: Marker, 1: pET-28a-toehold switch-amilCP plasmid digested byEcoR I and Hind Ⅲ restriction endonucleases, 2: pET-28a-toehold switch-amilCP plasmid, 3: pET-28a-toehold switch-mRFP plasmid digested by EcoRⅠ and Hind Ⅲ restriction endonucleases, 4: pET-28a-toehold switch-mRFP plasmid, 5: pET-28a-toehold switch-amilGFP plasmid digested by EcoRⅠ and Hind Ⅲ restriction endonucleases, 6: pET-28a-toehold switch-amilGFP plasmid.
Toehold switch plasmids work well for expressing reporter proteins in BL21 strain
The 3 toehold switch plasmids (pET-28a-toehold switch-amilCP, pET-28a-toehold switch-mRFP,andpET-28a-toehold switch-amilGFP) and corresponding miRNA were transfected into BL21 strain, respectively. The reporter proteins were expressed successfully, indicating that these toehold switch can interact with miR-34a-5p, miR-221-3p and let-7d-3p, respectively. (Plate 3 in Fig 2-4).
Fig.2 Expression of amilCP protein in BL21 strain.
1: Positive control of BL21 strain containing pET-28a-amilCP plasmid. 2: BL21 strain transfected only with pET-28a- toehold switch-amilCP plasmid, 3: BL21 strain transfected with pET-28a-toehold switch-amilCP plasmid and miR-34a-5p.
Fig.3 Expression of mRFP protein in BL21 strain.
1: Positive control of BL21 strain containing pET-28a-mRFP plasmid. 2: BL21 strain transfected only with pET-28a- toehold switch-mRFP plasmid, 3: BL21 strain transfected with pET-28a-toehold switch-mRFP plasmid and miR-221-3p.
Fig.4 Expression of amilGFP protein in BL21 strain.
1: Positive control of BL21 strain containing pET-28a-amilGFP plasmid. 2: BL21 strain transfected only with pET-28a- toehold switch-amilGFP plasmid, 3: BL21 strain transfected with pET-28a-toehold switch-amilGFP plasmid and let-7d-3p.
Obtain the best culture conditions of BL21 strains with toehold switch plasmids and miRNA transfection
We optimized BL21 strains culture conditions with trigger miRNAs (miR-34a-5p, miR-221-3p and let-7d-3p) for yielding more reporter protein production to increase the sensitivity of miRNA detection (Fig. 5-8).
Fig.5 The effect of pH value of culture media on the expression of reporter proteins in BL21 strains with toehold switch plasmids transfected with their corresponding miRNAs.
(A) BL21 strain with pET-28a-toehold switch-amilCP transfected with miR-34a-5p, (B) BL21 strain with pET-28a-toehold switch-mRFP transfected with miR-221-3p, (C) BL21 strain with pET-28a-toehold switch-amilGFP transfected with let-7d-3p. 1: pH6, 2: pH6.4, 3: pH6.8, 4: pH7.0, 5: pH7.2, 6: pH8.0, 7: pH8.4.
Fig.6 The effect of temperature on the expression of reporter proteins in BL21 strains with toehold switch plasmids transfected with their corresponding miRNAs.
(A) BL21 strain with pET-28a-toehold switch-amilCP transfected with miR-34a-5p, (B) BL21 strain with pET-28a-toehold switch-mRFP transfected with miR-221-3p, (C) BL21 strain with pET-28a-toehold switch-amilGFP transfected with let-7d-3p. 1: 27℃, 2: 29℃, 3: 31℃, 4: 33℃, 5: 35℃, 6:37℃, 7: 39℃.
Fig.7 The effect of fermentation time on the expression of reporter proteins in BL21 strains with toehold switch plasmids transfected with their corresponding miRNAs.
(A) BL21 strain with pET-28a-toehold switch-amilCP transfected with miR-34a-5p, (B) BL21 strain with pET-28a-toehold switch-mRFP transfected with miR-221-3p, (C) BL21 strain with pET-28a-toehold switch-amilGFP transfected with let-7d-3p. 1: 8h, 2: 10h, 3: 12h, 4: 14h, 5: 16h, 6: 18h, 7: 20h.
Fig.8 The effect of miRNA concentration on the expression of reporter proteins in BL21 strains with toehold switch plasmids transfected with their corresponding miRNAs.
(A) BL21 strain with pET-28a-toehold switch-amilCP transfected with miR-34a-5p, (B) BL21 strain with pET-28a-toehold switch-mRFP transfected with miR-221-3p, (C) BL21 strain with pET-28a-toehold switch-amilGFP transfected with let-7d-3p. 1: 0.1uM, 2: 0.25uM, 3: 0.5uM, 4: 0.75uM, 5: 1uM, 6:1.5uM, 7: 2uM.
The optimization experiment results indicated that pH7.2, 37°C, fermentation 18h, and 1.5uM miRNA are the best culture conditions for higher reporter protein production in E. coli BL21 strains.
Obtain the best reaction conditions for cell free expression system
In order to obtain sensitive and fast detection effects, the reaction conditions for expressing reporter proteins in cell free expression system were optimized under different temperature, rotation rate and reaction time. We took BL21 strain transfected with pET-28a-toehold switch-amilCP plasmid and its corresponding cell free expression system as an example to perform optimization experiments.
We obtain the best reaction conditions: the best temperature is 30 °C, 0 rpm is chosen considering its cost, and 1h is chosen as the best reaction time (Fig.9-11).
Fig.9 The optimization of temperature for the reporter protein expression in cell free system.
Fig.10 The optimization of rotation rate for the reporter protein expression in cell free system.
Fig.11 The optimization of reaction time for the reporter protein expression in cell free system.
Reporter gene expression on paper strip sensor based on the cell free system successfully
After the filter paper was treated, a certain amount of miRNA was dropped onto the paper strip sensor containing its corresponding toehold switch plasmid, the protein expression was successfully activated under the static conditions at 30°C for 1h (Fig.12).
Fig.12 Reporter gene expression triggered by different concentration of miRNA on the paper strip sensor based on the cell free reaction system.
(A) Expression of reporter gene amilCP triggered by different concentration of miR-34a-5p, (B) Expression of reporter gene mRFP triggered by different concentration of miR-221-3p, (C) Expression of reporter gene amilGFP triggered by different concentration of let-7d-3p.
3. Develop a paper strip sensor based on cell free system to detect miRNA through galactosidase reaction for MDD diagnosis
In order to increase the detection sensitivity, the reporter gene amilCP in the toehold switch plasmid was replaced with LacZ gene, expecting to amplify the reaction signal by the enzyme reaction that X-gal is cleaved to produce 5-bromo-4-chloro Indigo (blue color) catalyzed by β-galactosidase.
Obtain pET-28a-toehold switch-LacZ plasmid
Based on the flanking sequences of amilCP in pET-28a-toehold switch-amilCP plasmid, the amilCP gene was replaced with LacZ gene (3075bp) at EcoRⅠandHind Ⅲ cloning sites. pET-28a-toehold switch-lacZ was constructed successfully (Fig.13).
Fig.13 Identification of pET-28a-toehold switch-LacZ plasmid.
M: Marker, 1: The plasmid of pET-28a-toehold switch-LacZ, 2: The pET-28a-toehold switch-LacZ plasmid was digested by EcoRⅠ and Hind Ⅲ restriction endonuclease, 3: The LacZ gene amplified by PCR method.
Obtain BL21 strain with LacZ gene deletion
By homologous recombination, kanamycin resistant BL21 mutant strain with LacZ gene deletion (BL21DLacZ) was obtained successfully (Fig.14,15).
Fig.14 The PCR result of Kanamycin gene using pET-28a vector as a template.
This PCR product of kanamycin gene was flanked with the start and end sequences of LacZ gene. M: Marker, 1: Kanamycin gene amplified by PCR method.
Fig.15 The PCR results of Kanamycin and LacZ genes in kanamycin resistant BL21DLacZ strain.
M: Marker, 1: Kanamycin gene amplified by PCR method. 2. No band of LacZ gene amplified by PCR method.
Obtain a paper strip sensor for detecting miRNA using β-galactosidase reaction system
In order to obtain sensitive and fast detection effects, the reaction conditions under which X-gal is cleaved to produce 5-bromo-4-chloro Indigo (blue color) catalyzed by β-galactosidase in the cell free expression system from BL21DLacZ strain were optimized under different temperature, reaction time and miRNA concentration.
The best optimization results showed that the best temperature was 30°C, and 1h was chosen as the best reaction time (Fig.16,17). For miR-34a-5p target sensor, the lowest limit of visible color development is 500fM (Fig.18).
Fig.16 The optimization of temperature for reactions catalyzed by β-galactosidase in cell free system.
(A): OD570 value absorbed by the blue product, (B): Photograph of paper strip biosensor reaction in cell free system.
Fig.17 The optimization of reaction time for β-galactosidase catalyzing reaction in cell free system.
(A): OD570 value absorbed by the blue product, (B): Photograph of paper strip biosensor reaction in cell free system.
Fig.18 The optimization of miR-34a-5p concentration for β-galactosidase catalyzing reaction after 1h.
(A): OD570 value absorbed by the blue product, (B): Photograph of paper strip biosensor reaction in cell free system.
4. Design a small device for analyzing the results from paper strip sensor
In order to facilitate and quickly obtain diagnostic tests' results, we created a small device for it (Fig.19). There is a window on the side of the device where two paper strips can be placed. One paper strip is for reference and the other is for sample detection, which is convenient for qualitative and quantitative analysis.
There is a cell phone rack on the device, which can be retracted to facilitate the placement of smartphones of different sizes and specifications. The smartphone is used for acquiring image. The median value of the paper strip can be analyzed. Considering control reactions, the app calculates whether a contamination is present.
Fig.19 3D-printed device, filter, and paper strip sensor
5. Conclusion
We established a paper strip biosensor used for a detector to diagnose MDD by detecting miR-34a-5p, miR-221-3p and let-7d-3p. When using the chromoproteins as reporters, the lowest detecting concentration of target miRNAs is 10 ng/μL, 15 ng/μL and 15 ng/μL, respectively. When using β-galactosidase as a reporter, the lowest detecting concentration of target miR-34a-5p is 500fM, which increased more sensitivity.
This simple and convenient detector with filter paper strip biosensor is a powerful supplement to the current diagnosis of MDD which depends on clinical symptoms and self-assessment scale. This detector is very beneficial to the objective evaluation of MDD diagnosis. And people can use it to diagnose for himself even at home for privacy protection due to its simplicity and portability.