A Dual pH/O2 Sensing Film Based on Functionalized Electrospun Nanofibers for Real-Time Monitoring of Cellular Metabolism
Round 1
Reviewer 1 Report
Zhou and colleagues prepared a film-based dual-channel sensor that is responsive toward dissolved oxygen (DO) and pH. A porphyrin derivate was used to probe DO concentration and common fluorescein is employed in the pH sensing matrix. Benefiting from the electrospinning approach, the thin film exhibits good sensitivity and biocompatibility. Not limited to the applications in biotechnology, this approach can introduce an indicator on masks, which are also manufactured via electrospinning technique. Several minor concerns need to be addressed before appearing in the press.
- more properties of the thin film need to be characterized, such as thickness, elasticity, and hardness.
- it is recommended to include the absorption and emission spectra of FITC and PtTFPP together, and demonstrate the minimal FRET effect. In fact, the DO sensor is a phosphorescent probe and the pH sensor is a fluorescent probe, and the emission time domain can well separate these two signals.
- it is suggested to have discussions about quality control, in an effort to eliminate the artificial effect on probe doses varied from batch to batch.
Author Response
Response to Reviewer 1 Comments
General Comments: Zhou and colleagues prepared a film-based dual-channel sensor that is responsive toward dissolved oxygen (DO) and pH. A porphyrin derivate was used to probe DO concentration and common fluorescein is employed in the pH sensing matrix. Benefiting from the electrospinning approach, the thin film exhibits good sensitivity and biocompatibility. Not limited to the applications in biotechnology, this approach can introduce an indicator on masks, which are also manufactured via electrospinning technique. Several minor concerns need to be addressed before appearing in the press.
Comment 1: More properties of the thin film need to be characterized, such as thickness, elasticity, and hardness.
Response:Thank you for your pointing out the missing of important details. According to your suggestion, we have supplemented the cross-section characterization experimentally. The thickness was characterized by SEM cross-section morphology as shown in Figure S1. The thickness of F1 and F2 layer is about 5 µm and 15 µm, respectively. The following statement was added in Page 6, Line 207~210. The statement “Other than the surface appearance, Figure S1 shows the SEM images of the cross-section morphologies of F3 film, whereby the two-layer structure can be clearly detected. The thickness of F1 and F2 layer is about 5 µm and 15 µm, respectively.” was added.
Figure S1: SEM images of the film cross-section displaying the thickness of the two-layer membrane.
For the elasticity property, a stress-strain curved of F1 was tested by a tensile tester as shown in Figure S2. PCL is famous for its excellent tensile properties. The films modified by PCL have good flexibility. Page 6, Line 214-217. “Furthermore, stress-strain curves of F1 membrane was studied. The composite materials of CA and PCL nanofibers showed a high flexibility, the elongation at break was up to 148% (Figure S2). This property can broaden the application of the sensing films in other fields, such as the introduction of indicators on masks.”
Figure S2: The stress-strain behavior of F1 film.
Our films were made of flexible materials, so here we use a folding method to test the effect of mechanical deformation on the film sensing performance, as indicated in Figure S7. The statement “The resistance of the sensing film to mechanical deformation was also been explored (Figure S7). The sensing performance of the film barely changed after 10 times’ folding. This also demonstrated the stability of the film.” was added in Page 9, Line 303-305.
Figure S7: Response of the F1 sensing films before and after 10 times’ folding.
Comment 2: It is recommended to include the absorption and emission spectra of FITC and PtTFPP together, and demonstrate the minimal FRET effect. In fact, the DO sensor is a phosphorescent probe and the pH sensor is a fluorescent probe, and the emission time domain can well separate these two signals.
Response: We thank you very much for your important advice and detailed consideration. We are very sorry for the unclear statements in the manuscript. According to your suggestions, we added the following experiment. Emission spectrum of FITC and absorption spectrum of PTTFPP are shown in the Figure.S5. The relative descriptions were also added in revised manuscript. Page 9, Line 286~290. “Emission spectrum of FITC and absorption spectrum of PtTFPP are shown in Figure S5. Their spectra have overlap, but they are far away from each other in the two-layer film system, failing to reach the standard of the distance less than 10 nm. Therefore, there is almost no FRET phenomenon between them, which can be ignored.”
Your suggestion that the different emission time of phosphorescent and fluorescent probes is a good suggestion for signal separation. We will take this into consideration in the follow-up study.
Figure. S5: The absorption spectra of PtTFPP and emission spectra of FITC.
Comment 3: It is suggested to have discussions about quality control, in an effort to eliminate the artificial effect on probe doses varied from batch to batch.
Response: Thank you very much for your valuable suggestion. In fact, we've made a lot of efforts to control for variables. In terms of sample preparation, the concentration of electrospinning solution and probe ratio still keep consistent. During the process of machine operation, the electrospinning voltage and time, the distance between the needle and the receiving plate, and the injection speed remain constant. To ensure uniform film thickness, the needle was sprayed at a uniform speed. Most importantly, after cutting the film samples into a specific shape, we will first place them in a 96-well plate, test the fluorescence and phosphorescence intensity with a microplate reader, and select the film sample with the same intensity for subsequent experiments, so as to eliminate human interference as much as possible.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Authors have investigated thoroughly and written very well. Clean and neat figures, Literature was adequate, structure of the article is well kept.
This article can be accepted in its present form for publication in Molecules, rest leave it to authors and editor towards further improvement.
Author Response
Response to Reviewer 2 Comments
General Comments: Authors have investigated thoroughly and written very well. Clean and neat figures, Literature was adequate, structure of the article is well kept. This article can be accepted in its present form for publication in Molecules, rest leave it to authors and editor towards further improvement.
Response: Special thanks to you for your positive comments.
Author Response File: Author Response.docx
Reviewer 3 Report
A real-time pH/O2 sensing film was reported by electrospinning of cellulose acetate (CA) & poly(ε-caprolactone) (PCL) nanofiber membrane blended with platinum (II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTFPP) as the Oxygen sensing and nanofiber membrane of chitosan (CS) coupled with fluorescein 5-isothiocyanate (FITC) as the pH sensing. The supporting materials (CS, PCL and CA) were chosen for their biodegradability properties. The quenching of F3 was pH dependent between 3-9 and dissolved oxygen up to 40 mg/L affected the quenching linearly. Authors demonstrated that the nanofiber based material could determine oxygen and pH at better sensitivity than other similar sensors. The sensor was tested with a mouse cell line (3T3) and 1 bacterial cell (E. coli) and in vitro biocompability was determined by MTT test. I think this is an original study and it is interesting for researcher using live cell measurement of oxygen and pH. A minor point is that Figure 2C is the SEM figure for F3, but it is not referred in the text.
Author Response
Response to Reviewer 3 Comments
General Comments:A real-time pH/O2 sensing film was reported by electrospinning of cellulose acetate (CA) & poly(ε-caprolactone) (PCL) nanofiber membrane blended with platinum (II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTFPP) as the Oxygen sensing and nanofiber membrane of chitosan (CS) coupled with fluorescein 5-isothiocyanate (FITC) as the pH sensing. The supporting materials (CS, PCL and CA) were chosen for their biodegradability properties. The quenching of F3 was pH dependent between 3-9 and dissolved oxygen up to 40 mg/L affected the quenching linearly. Authors demonstrated that the nanofiber based material could determine oxygen and pH at better sensitivity than other similar sensors. The sensor was tested with a mouse cell line (3T3) and 1 bacterial cell (E. coli) and in vitro biocompability was determined by MTT test. I think this is an original study and it is interesting for researcher using live cell measurement of oxygen and pH. A minor point is that Figure 2C is the SEM figure for F3, but it is not referred in the text.
Response: Thanks for your nice comments. The relative descriptions were added in revised manuscript in Page 6, Line 205-206. The detailed corrections are listed below.
The statement “Eventually, we obtained a two-layer electrospun membrane F3, the morphology of F3 was shown in Figure 2c.”
