Impedance Sensing Platform for Detection of the Food Pathogen Listeria monocytogenes

Round 1

Reviewer 1 Report

In this work, the authors presented an EIS biochip with a microfluidic platform to detect L. monocytogenes in artificially contaminated milk samples. The work described is of interest, however there are a few points that need to be addressed.

In the introduction, the authors have described previous work and other biochips. However, they have not clearly mentioned why they technology/biochip is more useful. As a suggestion, they could highlight the point-of-care aspect of the device.

Although microfluidics plays an important part of this chip, but there is very little mention of the chamber design and why specific configuration was used. Please provide more detail. Also, it would be helpful to add a schematic of the microfluidic chambers, highlighting the dimensions, as well as the purpose. 

Please place the figures appropriately throughout the manuscript (along the citation of the figure in the text), not in one section. (alternatively, the figures should be placed at the very end of the manuscript (not in the middle before the discussion).

What was the inherent error range of pocketSTAT? Was this error in measurement taken into account while developing the calibration curve?

How do you account for increase in impedance in case of sensing S. enteric?

There are other chips that provide a lower LOD. How do the authors propose to improve the LOD for future implementation? 

what viscosity does the liquid sample has to have in order to improve sensor performance?

Authors should carefully proof-read the manuscript.

Author Response

We thank Reviewer 2 for his/her positive comments and we hope that now all the issues have been clarified by a point-to-point response. Modified portions of the manuscript are highlighted in red.

In the introduction, the authors have described previous work and other biochips. However, they have not clearly mentioned why they technology/biochip is more useful. As a suggestion, they could highlight the point-of-care aspect of the device.

In the revised version of the manuscript the aspects of portability and point-of-care feature of the microfluidic and sensing platform have been stressed in the introduction chapter and we thank the reviewer for the precious suggestions. “The biochip was connected to a portable potentiostat and to a notebook, which make the platform very suitable for on-field applications and. in particular it exploits the advantages of flow immunoassays as quantitative results, speed, reduced sample handling and costs. In the developed protocol of analysis there is no need for fluorescent tagging of reagents, no PCR amplification and no complex instrumentation or skilled operators. Our biochips for detection of milk contamination down to 5 CFU/ml represents a robust, portable platform for a fast and low-cost analysis which is very suitable for use in food factories since it can allow to test, at the same time, different batches of products for their contamination levels. Definitely, due to the high flexibility, predisposition to further miniaturization, possibility to improve the number of analytes and the ease of use, the developed device is competitive with currently used methods and This platform  can be used as point-of-care device for clinical and food diagnostics, as well as for biosecurity purposes.” (page 3)

Although microfluidics plays an important part of this chip, but there is very little mention of the chamber design and why specific configuration was used. Please provide more detail. Also, it would be helpful to add a schematic of the microfluidic chambers, highlighting the dimensions, as well as the purpose. 

As required, a detailed description of the microfluidic module has been added in the revised version of the manuscript: “The developed tool consists of an array of interdigited electrodes aligned at the bottom of a network of four microchambers and related microchannels. Microelectrodes in this case were functionalized with antibodies specific for L. monocytogenes. The network provides the possibility to separately fill the different chambers (to allow multiple functionalization or to inject different samples from peripheral holes) or in turn to use a central inlet common to the four chambers, in order to detect different analytes from a single sample. In our experiment a chamber was used for the detection of L. monocytogenes and the other one as negative control.” (page 3, Introduction section) and “The microfluidic module is obtained from a SU8 hard master by PDMS replica molding. The volume of each microchamber is of around 15 µl (3 mm diameter and 0.5 mm height) and the architecture of the network provides a common central inlet and four peripheral holes that can be used in turn to independently functionalize each chamber or to inject a sample to be tested in quadruplicate. In order to completely fill the chambers considering microchannels, a volume of 80 µl is enough. The presence of the microfluidic module allows a high degree of reproducibility of experiments and to save large quantity of reagents and samples.” (page 4, Materials and Methods section). A new figure with a schematic representation of microfluidic and sensing module has been also inserted as Figure 1.

Please place the figures appropriately throughout the manuscript (along the citation of the figure in the text), not in one section. (alternatively, the figures should be placed at the very end of the manuscript (not in the middle before the discussion).

As required from Reviewer 1 figures have been rearranged in order to follow their mention through the manuscript. 

What was the inherent error range of pocketSTAT? Was this error in measurement taken into account while developing the calibration curve?

Calibration curves derive from the raw impedance measurements collected by pocketSTAT. Measurements of the same samples have been performed using different electrodes giving rise to the reported error bar.    

How do you account for increase in impedance in case of sensing S. enteric?

S. enterica induces a low impedance increase over the antibody baseline. This increase may be due to an unspecific adsorption of matrix components over the sensing molecular layer which in turn may be attributed to culture medium or milk residuals. 

There are other chips that provide a lower LOD. How do the authors propose to improve the LOD for future implementation? 

Even in literature some other examples are present, our biochip has advantages of portability, low costs and ease of use that makes it competitive with other detection tools. Improvements in LOD may require the addiction of tools for signal enhancement as for example the labelling of cells with metal nanoparticles able to recognize species-specific surface antigens. This would increase the sensibility of the device but will be detrimental for costs and will require additional steps for sample preparation, which would affect portability and probably require the presence of specialized personnel to allow the correct use of the platform. 

what viscosity does the liquid sample has to have in order to improve sensor performance?

Viscosity is a parameter which is strictly related with the sample. Viscosity of the sample may affect the biorecognition events but can be bypassed by optimizing a dedicated buffer to dilute the sample and making antigens accessible to the antibody layer. A similar situation has been issued in other works we published dealing with a layer of antibodies immobilized on the surface of electrodes and antigens to be collected from viscous biological samples or vice versa. In a first case (Towards pancreatic cancer diagnosis using EIS biochips. Lab Chip. 2013; 13(4):730-4. doi: 10.1039/c2lc41127j) serum samples were diluted in Phosphate Buffer Solution (1:100), in another work we used vaginal fluid samples collected by diluting swabs in physiological solution or resuspended in sterile PBS (Simultaneous detection of multiple lower genital tract pathogens by an impedimetric immunochip. Biosensors and Bioelectronics. 2016 79, 15, 9-14; doi.org/10.1016/j.bios.2015.11.100). In another case (unpublished data) we developed a buffer with PBS, Tween 20 surfactant and BSA (bovine serum albumine) to work with 75% serum. In the case of milk, used to mimic real food samples contaminated by Listeria monocytogenes, as we highlighted in Discussion section of the manuscript, probably casein acts as a natural passivation agent and in laboratory protocols of sensing methods it is often added to improve the sensibility of the assay. 

Authors should carefully proof-read the manuscript.

A general revision of the manuscript for language and typos has been performed. 

Author Response File: Author Response.pdf

Reviewer 2 Report

Please refer to the attached file for the comments.

This manuscript presents an impedance-based biosensor in microchannels for detection of L. monocytogenes in food. The device looks novel and the authors demonstrate the specificity, detection range, and limit of detection of the assay with standard and spiked samples. The signals respond with different concentrations very well but further details and illustration of the figures should be included in the revised version.

1.    What is the material used for fabrication of the microchannel? A description is needed in the microfabrication part.  Zoom-in images showing the configuration of electrodes and microchannels are necessary in Fig.1.

2.    Should the authors explain in more details about the semicircle shape of the curves and why some of the curves went up after one cycle?

3.    Are those error bars from single samples with multiple measurements at different time points. or are they obtained from measuring different samples?

4.    The calibration curve does not look like a linear, what is the R value?

5.    Could the authors compare the limit of detection achieved in this study with previously published ones for detection of L. monocytogenes? The method of estimating the LOD could be wrong based on the calibration curve and the data present in fig. 2-4. Please check.

Comments for author File: Comments.pdf

Author Response

We thank Reviewer 2 for his/her positive comments and we hope that now all the issues have been clarified by a point-to-point response. Modified portions of the manuscript are highlighted in red.

In the introduction, the authors have described previous work and other biochips. However, they have not clearly mentioned why they technology/biochip is more useful. As a suggestion, they could highlight the point-of-care aspect of the device.

In the revised version of the manuscript the aspects of portability and point-of-care feature of the microfluidic and sensing platform have been stressed in the introduction chapter and we thank the reviewer for the precious suggestions. “The biochip was connected to a portable potentiostat and to a notebook, which make the platform very suitable for on-field applications and. in particular it exploits the advantages of flow immunoassays as quantitative results, speed, reduced sample handling and costs. In the developed protocol of analysis there is no need for fluorescent tagging of reagents, no PCR amplification and no complex instrumentation or skilled operators. Our biochips for detection of milk contamination down to 5 CFU/ml represents a robust, portable platform for a fast and low-cost analysis which is very suitable for use in food factories since it can allow to test, at the same time, different batches of products for their contamination levels. Definitely, due to the high flexibility, predisposition to further miniaturization, possibility to improve the number of analytes and the ease of use, the developed device is competitive with currently used methods and This platform  can be used as point-of-care device for clinical and food diagnostics, as well as for biosecurity purposes.” (page 3)

Although microfluidics plays an important part of this chip, but there is very little mention of the chamber design and why specific configuration was used. Please provide more detail. Also, it would be helpful to add a schematic of the microfluidic chambers, highlighting the dimensions, as well as the purpose. 

As required, a detailed description of the microfluidic module has been added in the revised version of the manuscript: “The developed tool consists of an array of interdigited electrodes aligned at the bottom of a network of four microchambers and related microchannels. Microelectrodes in this case were functionalized with antibodies specific for L. monocytogenes. The network provides the possibility to separately fill the different chambers (to allow multiple functionalization or to inject different samples from peripheral holes) or in turn to use a central inlet common to the four chambers, in order to detect different analytes from a single sample. In our experiment a chamber was used for the detection of L. monocytogenes and the other one as negative control.” (page 3, Introduction section) and “The microfluidic module is obtained from a SU8 hard master by PDMS replica molding. The volume of each microchamber is of around 15 µl (3 mm diameter and 0.5 mm height) and the architecture of the network provides a common central inlet and four peripheral holes that can be used in turn to independently functionalize each chamber or to inject a sample to be tested in quadruplicate. In order to completely fill the chambers considering microchannels, a volume of 80 µl is enough. The presence of the microfluidic module allows a high degree of reproducibility of experiments and to save large quantity of reagents and samples.” (page 4, Materials and Methods section). A new figure with a schematic representation of microfluidic and sensing module has been also inserted as Figure 1.

Please place the figures appropriately throughout the manuscript (along the citation of the figure in the text), not in one section. (alternatively, the figures should be placed at the very end of the manuscript (not in the middle before the discussion).

As required from Reviewer 1 figures have been rearranged in order to follow their mention through the manuscript. 

What was the inherent error range of pocketSTAT? Was this error in measurement taken into account while developing the calibration curve?

Calibration curves derive from the raw impedance measurements collected by pocketSTAT. Measurements of the same samples have been performed using different electrodes giving rise to the reported error bar.    

How do you account for increase in impedance in case of sensing S. enteric?

S. enterica induces a low impedance increase over the antibody baseline. This increase may be due to an unspecific adsorption of matrix components over the sensing molecular layer which in turn may be attributed to culture medium or milk residuals. 

There are other chips that provide a lower LOD. How do the authors propose to improve the LOD for future implementation? 

Even in literature some other examples are present, our biochip has advantages of portability, low costs and ease of use that makes it competitive with other detection tools. Improvements in LOD may require the addiction of tools for signal enhancement as for example the labelling of cells with metal nanoparticles able to recognize species-specific surface antigens. This would increase the sensibility of the device but will be detrimental for costs and will require additional steps for sample preparation, which would affect portability and probably require the presence of specialized personnel to allow the correct use of the platform. 

what viscosity does the liquid sample has to have in order to improve sensor performance?

Viscosity is a parameter which is strictly related with the sample. Viscosity of the sample may affect the biorecognition events but can be bypassed by optimizing a dedicated buffer to dilute the sample and making antigens accessible to the antibody layer. A similar situation has been issued in other works we published dealing with a layer of antibodies immobilized on the surface of electrodes and antigens to be collected from viscous biological samples or vice versa. In a first case (Towards pancreatic cancer diagnosis using EIS biochips. Lab Chip. 2013; 13(4):730-4. doi: 10.1039/c2lc41127j) serum samples were diluted in Phosphate Buffer Solution (1:100), in another work we used vaginal fluid samples collected by diluting swabs in physiological solution or resuspended in sterile PBS (Simultaneous detection of multiple lower genital tract pathogens by an impedimetric immunochip. Biosensors and Bioelectronics. 2016 79, 15, 9-14; doi.org/10.1016/j.bios.2015.11.100). In another case (unpublished data) we developed a buffer with PBS, Tween 20 surfactant and BSA (bovine serum albumine) to work with 75% serum. In the case of milk, used to mimic real food samples contaminated by Listeria monocytogenes, as we highlighted in Discussion section of the manuscript, probably casein acts as a natural passivation agent and in laboratory protocols of sensing methods it is often added to improve the sensibility of the assay. 

Authors should carefully proof-read the manuscript.

A general revision of the manuscript for language and typos has been performed.