Review Reports - Detecting EGFR Gene Mutations on a Nanobioarray Chip

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript describes the detection of mutations relevant to the EGFR gene that occur in exon 20 (T790M) and exon 21 (L858R and L861Q) from genomic DNA samples in a 16-channel chip. As mutated EGFR is the target of a therapy for non-small cell lung cancer (NSCLC) using tyrosine kinase inhibitors (TKIs) as treatment drugs, the accurate qualification may guide the therapeutics before and during treatment. The manuscript was not well written and should be carefully revised before further evaluation:

1) First of all, the work looks like an experimental report, instead of a scientific paper. For example, the authors used different washing procedures. However, they do not explain the reasons for different results obtained.

2) A clear Scheme should be provided, with complete hybridization details.

3) As the authors used cy5 fluorescent label, the further quantitative comparison are required, according to differentiation ratio (DR). Moreover, have the authors considered the error ranges of DR?

4) Figure 3, how to optimize the working temperature?

5) For Au NPs wash, what about the reason to choose NPs with a size of 5 nm? What about the protecting agent of NPs (maybe citrate)? Since 8 nM stabilizing DNA was simultaneously used, have the authors investigated the effect of only stabilizing DNA as washing buffer?

6) The detection limit (copies) of the mutation should be provided.

7) More clinical samples are necessary to enhance the applicability of the protocol.

8) Generally, more references are required. Note that most works cited are published before 2020 , the recently published works should be included.

Author Response

The reasons are given in the results and discussion section as:

lines 137-139: The binding intensity towards the WT probe is quite weak, and this can be attributed to the secondary structures found in the 60-mer ssDNA, and in the probe.

lines 152-155: This indicates the use of both elevation in hybridization temperature and a subsequent dynamic AuNP wash has achieved satisfactory differentiation of the two point mutations in exon 21.

lines 190-192: These challenges have been resolved by the use of AuNP wash, which invokes the kinetic consideration, in addition to the equilibrium one.

2) A clear Scheme should be provided, with complete hybridization details

Fig 1 is clear in depicting the hybridization by targets onto immobilized vertical probe lanes, followed by SA-Cy5 fluorescence labeling. The hybridization details are provided on lines 110-113 as: And then biotin-labeled target solutions (volume of 1 mL) were introduced through the channels. The targets hybridized at room temperature to the immobilized probe for 1 h. Post-hybridization, either a buffer wash or AuNP wash (5 nm) was conducted.

3) As the authors used cy5 fluorescent label, the further quantitative comparison are required, according to differentiation ratio (DR). Moreover, have the authors considered the error ranges of DR?

The errors in DR are ±0.2.

4) Figure 3, how to optimize the working temperature?

The temperature of 47 °C has been optimized in previous reports, refs 14, 16.

The conditions for AuNP wash, i.e. particle size, stabilizing DNA, were optimized previously, as reported in Refs 14, 16.

6) The detection limit (copies) of the mutation should be provided.

The PCR products used in Fig 7 (volume of 1 mL) was 25 nM or 25e-9 x 1 e-6 = 25 femtomoles or 1.5 e10 copies.

7) More clinical samples are necessary to enhance the applicability of the protocol.

The clinical samples were the genomic DNAs purchased from Horizon Discovery: HD709 (50 ng/µL) for the wild-type EGFR gene, and HD802 (50 ng/µL) for the mutated one, see lines 83-85.

8) Generally, more references are required. Note that most works cited are published before 2020 , the recently published works should be included.

New post-2020 references are added.

He, C., Wei, C., Wen, J. et al. Comprehensive analysis of NGS and ARMS-PCR for detecting EGFR mutations based on 4467 cases of NSCLC patients. J Cancer Res Clin Oncol. 2022, 148, 321–330.

Rebekka Van Hoof, Michal Szymonik, Stefanos K. Nomidis, Karen Hollanders, An Jacobs, Inge Nelissen, Patrick Wagner, Jef Hooyberghs, "Depletion of wild-type target enhances the hybridization-based sensitivity of low-abundant mutation detection by reference capture probes," Sensors and Actuators B: Chemical, 368, 2022, 132175.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study presents a rapid, low-cost nanobioarray-based DNA hybridization method for detecting clinically relevant EGFR point mutations (T790M, L858R, and L861Q) associated with tyrosine kinase inhibitor resistance or sensitivity in non-small cell lung cancer. By combining optimized hybridization conditions with a gold nanoparticle–assisted wash, the authors demonstrate clear discrimination between wild-type and mutant EGFR sequences using both synthetic targets and PCR-amplified genomic DNA. Overall, the approach offers a faster alternative to conventional DNA sequencing for routine EGFR mutation screening and has potential clinical utility. However, while the topic is important and timely in the field of oncology, several sections of the manuscript require further revision and clarification before it can be considered for publication in Biomedicines.

The study primarily uses synthetic oligonucleotides and commercial genomic DNA. Have the authors considered validating the method using clinical NSCLC patient samples to better demonstrate its real-world applicability?
Please clarify the analytical sensitivity of the assay. What is the minimum mutant allele frequency that can be reliably detected?
How does the assay perform in the presence of closely related EGFR mutations or non-target SNPs? Additional data or discussion regarding potential cross-hybridization would strengthen confidence in the assay’s specificity.
Please provide information on inter-assay and intra-assay variability. Were experiments repeated on different days or with different nanobioarray chip batches, and how consistent were the resulting differentiation ratios?
The choice of hybridization temperature (47 °C) and AuNP wash conditions appears to be critical for assay performance. Could the authors clarify how these parameters were optimized and whether the assay is robust to small variations in experimental conditions?
While a 16-channel nanobioarray chip is demonstrated, please discuss the potential scalability of the platform, particularly for multiplexed detection or higher-throughput clinical applications.
The manuscript states that the method can be completed within one day. A more detailed breakdown of the workflow steps and associated time requirements would help readers better assess the feasibility of clinical implementation.

Author Response

The study primarily uses synthetic oligonucleotides and commercial genomic DNA. Have the authors considered validating the method using clinical NSCLC patient samples to better demonstrate its real-world applicability?

The clinical samples were the genomic DNAs purchased from Horizon Discovery: HD709 (50 ng/µL) for the wild-type EGFR gene, and HD802 (50 ng/µL) for the mutated one, see lines 83-85.

Please clarify the analytical sensitivity of the assay. What is the minimum mutant allele frequency that can be reliably detected?

The PCR products used in Fig 7 (volume of 1 mL) was 25 nM or 25e-9 x 1 e-6 = 25 femtomoles or 1.5 e10 copies.

According to the information provided by Horizon Discovery, the allele frequencies of L861Q, L858R and T790M in HD802 are 12.5%

How does the assay perform in the presence of closely related EGFR mutations or non-target SNPs? Additional data or discussion regarding potential cross-hybridization would strengthen confidence in the assay’s specificity.

The assay results for wild-type and mutated EGFR are depicted in Fig 7.

Please provide information on inter-assay and intra-assay variability. Were experiments repeated on different days or with different nanobioarray chip batches, and how consistent were the resulting differentiation ratios?

The experiments were repeated at least 3 times. The errors in DR are ±0.2.

The choice of hybridization temperature (47 °C) and AuNP wash conditions appears to be critical for assay performance. Could the authors clarify how these parameters were optimized and whether the assay is robust to small variations in experimental conditions?

The temperature of 47 °C has been optimized in previous reports, refs 14, 16.

While a 16-channel nanobioarray chip is demonstrated, please discuss the potential scalability of the platform, particularly for multiplexed detection or higher-throughput clinical applications.

The chip used here consisted of 16 channels. Other chips that contains 96 channels for high-throughput tests have been previously reported, see refs 12, 16.

The manuscript states that the method can be completed within one day. A more detailed breakdown of the workflow steps and associated time requirements would help readers better assess the feasibility of clinical implementation.

The workflow has been depicted in Fig 1, as well as in figures in previously reports, e.g. refs 11, 13, 14.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have no further comments now.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have satisfactorily revised the manuscript in accordance with the reviewers’ recommendations. As no further comments are needed, the reviewers have accepted the manuscript for publication in ฺBiomedicines.