Handheld Enzymatic Luminescent Biosensor for Rapid Detection of Heavy Metals in Water Samples
, Ivan A. Denisov 1,2,*,†, Vladimir V. Sorokin 2,†, Anton S. Yakimov 3, Elena N. Esimbekova 2,4 and Peter I. Belobrov 2Round 1
Reviewer 1 Report
First preliminary results with luminescent biosensors were presented. For a rapid publication, it is acceptable. However, what I miss are experiments with more concentrations of CuSO4 than the two presented. This would help to understand the proposed LOD of 2.5 mg/l. A comparison between the handheld and the GloMax® 20/20 luminometer was shown in Figure 3. Is it possible to address the causes of lower resolution of the SiPM handheld luminometer at higher intensities? In the conclusion, applications in other areas were proposed. However, by my opinion, it was too general. Could the authors please specify one or two examples, for which the handheld luminometer shows real advantages over commercial devices, especially because ofthe lower resolution described?
Specific comments
p.2 l.45: costs effective
p.2 l.67: promising
p.2 l.70: cheaper
p.3 l. 82: Merck
Figure 1: chip (2)
p.4 l.141: from opaque
Figure 2: too much information, it can be skipped and referred to table 1.
Author Response
Response to Reviewer 1 Comments
Point 1: First preliminary results with luminescent biosensors were presented. For a rapid publication, it is acceptable. However, what I miss are experiments with more concentrations of CuSO4 than the two presented. This would help to understand the proposed LOD of 2.5 mg/l.
Response 1: The detailed analysis of the sensitivity of microfluidic chips with a bioluminescent system to various types of toxins was made earlier in another paper (Denisov, Lukyanenko et.al. 2018). The laboratory luminometer GloMax 20/20 was used as a detector in those experiments, as well as microfluidic chips without an optimized composition of the bioluminescent system. In that work, it was shown that the LOD for chips is at the level of 3 μM. The optimization of the composition of microfluidic chips in this work allowed us to increase not only the intensity of the luminescence level, but also increase the sensitivity to toxic substances, as can be seen from Fig. 2 (now Fig.3). Based on this data and the data in Fig. 3 (now Fig.4), it was concluded that the detection limit for CuSO4 is 2.5 mg/l.
However, indeed, we do not provide a wider range of data on CuSO4 concentrations due to the fact that these points were not measured. In this work, we wanted to show the fundamental possibility of using microfluidic chips for working with a portable luminometer within the framework of the point-of-care concept.
Point 2: A comparison between the handheld and the GloMax® 20/20 luminometer was shown in Figure 3. Is it possible to address the causes of lower resolution of the SiPM handheld luminometer at higher intensities?
Response 2: As can be seen from the error bars at fig. 3 (now Fig.4) there is no significant difference between suggested luminometer and GloMax for the 18.7 mg/l. With less concentration and more light intensity this difference can be distinguished. Lower resolution is related to the size of the surface area of the photodetectors and different optical system for light condensation. The optical system of suggested device can be improved by using of anti-reflection coating for glass prism and better back mirror quality. However the sensitivity we demonstrated in the manuscript already showing possibility for using the portable device as an alternative for the field work. We improved the discussion for figure 3 (now Fig.4) in the manuscript to make it more clear.
Point 3: In the conclusion, applications in other areas were proposed. However, by my opinion, it was too general. Could the authors please specify one or two examples, for which the handheld luminometer shows real advantages over commercial devices, especially because of the lower resolution described?
Response 3: Portable size of the proposed biosensor allows for testing “at the place of demand”, for example, during field work, when it is not possible to take expensive laboratory equipment. This example was inserted into the conclusions section.
Point 4: Specific comments
p.2 l.45: costs effective
p.2 l.67: promising
p.2 l.70: cheaper
p.3 l. 82: Merck
Figure 1: chip (2)
p.4 l.141: from opaque
Figure 2: too much information, it can be skipped and referred to table 1.
Response 4: Thank you for indicating the typos and mistakes. We referred to the table in the figure caption. However we kept extra comments, because they allow better understanding of table and figure.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The work carried out by the authors on bioluminescence is not novel but still encouraging. From the paper it is understood that a lot of research component is missing throughout the work, hence need major revision. Following remarks should be addressed by the authors.
1. If the emphasis is on the development of the instrument, then all required details of the instrument should be included in it. It seems the parts and assemblies author got is developed/made elsewhere. Its better author should include a circuit block diagram and program algorithm while describing the instrument construction.
2. Why the work is performed for one enzyme and one metal ions?
3. The author should present the response to various other metal ions towards the sensitivity of the selected enzyme.
4. Does the author evaluated cross sensitivity? Is the used enzyme is sensitive to only to copper sulphates?
5. How many microfluidic chips were utilized for the one experiment? The author needs to comment on repeatability and reproducibility of the chips
6. Why CuSo4 range is selected from 3.7 to 18.7 mg/L? What is the detection limits of the developed sensor/ instrument?
Author Response
Response to Reviewer 2 Comments
The work carried out by the authors on bioluminescence is not novel but still encouraging. From the paper it is understood that a lot of research component is missing throughout the work, hence need major revision. Following remarks should be addressed by the authors.
Point 1: If the emphasis is on the development of the instrument, then all required details of the instrument should be included in it. It seems the parts and assemblies author got is developed/made elsewhere. Its better author should include a circuit block diagram and program algorithm while describing the instrument construction.
Response 1:Comparing with many journal articles, which present the devices concepts, we are confident, that it is not necessary to provide all the data including program code etc. We provide the models of the detector and amplifier which we are using. However, we agree that block diagram, which describes the principle of the device, is important here. We also added microcontrollers’ models to the materials section. The electrical and software engineers with sufficient experience can reproduce the device using provided block diagram.
Point 2:Why the work is performed for one enzyme and one metal ions?
Response 2:This work focuses on the proof-of-concept of the possibility of using a portable biosensor with disposable microfluidic chips for bioassay at the place of demand. The bienzyme bioluminescent bacteria system is used in microfluidic chips, because we have previously worked with this system and it was more accessible to test the concept. The use of other enzymatic systems in these microfluidic chips is also possible; however this was not the focus of our research.
Point 3:The author should present the response to various other metal ions towards the sensitivity of the selected enzyme. Does the author evaluated cross sensitivity? Is the used enzyme is sensitive to only to copper sulphates?
Response 3:We have previously published a paper, in which we described a more detailed sensitivity analysis of microfluidic chips with a bioluminescent system presented here to other types of toxins (Denisov, Lukyanenko et.al. 2018). In general, the bioluminescent system proposed as a biotest is sensitive to a wide class of toxic substances and, in addition to environmental biassay in water (Vetrova, Esimbekova et al., 2007; Esimbekova, Kondik et al., 2013), can be used in various fields:
1) Evaluation of the potential toxicity of carbon nanomaterials (Esimbekova, Nemtseva, et al., 2017)
2) Plant stress assessment (Kratasyuk, Esimbekova, Correll, et al., 2011)
3) Assessment of the toxicity of food additives (Esimbekova, Asanova, et al., 2017)
4) Determination of nonspecific endotoxicosis (Esimbekova, Kratasyuk, Abakumova, 1999)
Point 4:How many microfluidic chips were utilized for the one experiment? The author needs to comment on repeatability and reproducibility of the chips
Response 4:Microfluidic chips presented in the work are disposable. For each of the points in Fig. 3 at least 12 chips were used and a statistical error analysis was performed with a confidence interval of 95%. Corresponding explanations were included in the text in the section “Testing procedure” and highlighted in red.
Point 5:Why CuSo4 range is selected from 3.7 to 18.7 mg/L? What is the detection limits of the developed sensor/ instrument?
Response 5:The concentration of 3.7 mg/l is the EC50value, which was shown in our previous work (Denisov, Lukyanenko et.al. 2018). The concentration of 18.7 mg/l is the concentration at which almost complete inhibition of luminescence occurs, and was chosen for clarity.
The detection limit for developed instrument is around 2000 RLU due to noise/signal ratio assumptions. This value was not mentioned in the publication, because the exact detection limit of the developed portable luminometer was not investigated due to the absence of a calibrated reference source of photons. Therefore, a comparative analysis of the sensitivity of the device with a known laboratory luminometer was carried out using microfluidic chips as an “chemical reference”. Nevertheless, this question is indeed important and will be the subject of research in future publications.
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Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Response to reviewer is satisfactory
