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Detection of EGFR gene status via DNA electrochemical biosensor based on lambda exonuclease-assisted signal amplification.

Based on our previous work, we have constructed a new electrochemical biosensor to detect epidermal growth factor receptor (EGFR) gene mutation, which was a significant therapeutic effect predictor of target drugs for non-small cell lung cancer. In order to lower the detection limit to detect the small amount of EGFR gene status, we have employed lambda exonuclease (λ-Exo) to form a hybridization-digestion cycle. The reaction stages are depicted as follows: the target DNA hybridized with auxiliary DNA which had been modified with the λ-Exo recognition site; then, the double strands were cleaved by λ-Exo. The target DNA was released completely, and continued to hybridize with remaining auxiliary DNA, which formed a recycle for target reutilization. Finally, we detected the remaining auxiliary DNA to evaluate the amount or status of the EGFR gene. The reutilization of target DNA will help to achieve an enlarged signal with a small amount of target DNA, and the detection limit of the biosensor decreased down to 10 pM. Meanwhile, our assay can differentiate wild genes from the mutational gene of EGFR with excellent specificity. Our signal amplification method provides a research foundation for the detection system of the electrochemical biosensor by employing exonuclease, and impels the biosensor to be developed as a suitable method for EGFR detection in clinical applications.


Introduction
Lung cancer has a relatively high incidence and mortality. 1 Tyrosine kinase inhibitors targeting epidermal growth factor receptor (EGFR) exert a significant therapeutic effect on lung cancer. The status of the EGFR gene is an important therapeutic effect predictor for target drug treatments, which show greater efficiencies in patients with EGFR gene mutations. 2 The United States National Comprehensive Cancer Network (US NCCN) Guidelines recommend testing for EGFR mutations in patients with advanced lung cancer to guide the use of targeted therapies. Tumor tissue specimens used in conventional genetic testing are difficult to obtain from some patients because of the invasive nature of sample collection and the difficulties in retrieving specimens.
In contrast, body fluid specimens, such as blood and tissue fluids, can be obtained easily. But the low levels of tumor DNA present in these samples increase the difficulty of detection so that puts forward new challenges to detection methods. Therefore, it is urgent to develop a highly sensitive detection method to meet current clinical needs. excellent specificity and high cleavage activity for double-stranded DNA. 7 In our previous work, we employed λ-Exo to digest double-stranded to form single-stranded DNA with high efficiency. 8 Basing on sufficient familiarity with λ-Exo characteristics, we try to combine λ-Exo with DNA electrochemical biosensor to construct a novelty electrochemical biosensor.
We also optimized the detection system to further amplify the signals based on the observation that the electron transfer capacity is affected by the distance between the electroactive materials and electrode surface. The electron transfer capacity is affected by the distance between the electroactive materials and electrode surface to some extent, with a shorter distance facilitating the diffusion of electroactive materials to the electrode surface 9 and potentially generating a more substantial signal. In conventional methods, biotin is located away from the electrode surface on double-stranded DNA. The binding between biotins and streptavidin-horseradish peroxidase (HRP) catalyzes the production of electroactive materials, which must diffuse a distance equivalent to the length of the double-stranded DNA before arriving at the electrode surface. This study was conducted to develop a method to accelerate the rate of electron transfer and improve the detection of electrical signals by shortening the distance between the electroactive materials and electrode surface.
We previously developed an electrochemical biosensor for detecting EGFR mutations and differentiating between wild-type EGFR and mutant EGFR. 8 Additionally, we identified the mutation types by grouping analysis. 10 In this study, we combined the biosensor with λ-Exo assisted hybridization enzymatic-digestion

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DNA hybridization＆digestion
The target DNA and auxiliary DNA were mixed in a certain proportion ranging from 1:1 to 1:100 in 50 µL reaction volume containing 0.2 U/µL λ-Exo and reaction buffer dilution. The mixture was incubating at 37 ℃ for 30-150 min. After the reaction finished, λ-Exo was inactivated at 75 ℃ for 10 min according to the manual. The remaining auxiliary DNA was detected after incubating with capture and reporter probes.

Electrochemical biosensor detection
An electrochemical analyzer (CHI660C, Shanghai, China) was used for the amperometric and current-time curve experiments were connected to a conventional Au disk working electrode (2 mm diameter), an Ag/AgCl reference electrode, and a Pt wire auxiliary electrode. The BSA-based biosensor was fabricated according to a protocol developed previously. 11 The remaining auxiliary DNA was detected by biosensor follow the procedure in our previous study. The auxiliary DNA was mixed with the biotinylated reporter probe (20 nM) in the hybridization buffer, then the mixture was incubated with capture probe immobilized on the electrode surface. After streptavidin-HRP spreading on the electrode surface, the biosensor was extensively rinsed and subjected to electrochemical measurements. Amperometric detection was performed in the TMB substrate at 0 V, and the current was sampled at 100 s. Each experiment was repeated at least three times.

Working Principle
In this study, we employed the HEC cycle method for signal amplification. The λ-Exo was mixed with the target and auxiliary DNA strands in a reaction system.
Hybridization of the auxiliary DNA harboring the recognition and cleavage sites of In numerous recent studies, nuclease digestion was conducted on the electrode surface. Various factors including steric hindrance and electrostatic repulsion, as well as interactions between the active sites of enzymes and electrode surface, can reduce the rate of enzymatic digestion. 12,13 Exonucleases have several advantages in a liquid homogeneous system such as high digestion rate, high selectivity, and simplicity in DNA detection. Therefore, the HEC reaction cycle in this study was carried out in a liquid homogeneous medium, followed by the detection of auxiliary DNA to indirectly determine the amount of target DNA. 37 °C in a water bath for 1 h to detect the enzymatic digestion products via the time-current curve method. The results were compared to those of the enzyme-free group. The results ( Figure 3) revealed no difference in electrical signals with or without target DNA in the absence of λ-Exo. After adding λ-Exo, the electrical signals in the λ-Exo group were similar to those of the λ-Exo-free group in the absence of target DNA, which demonstrated that λ-Exo did not affect the working procedure of the detection system. However, the electrical signals decreased significantly in the presence of target DNA, indicating the involvement of λ-Exo in the HEC cycle with relatively higher activity levels in this homogeneous reaction system. λ-Exo began cleaving DNA in a 5′-to-3′ direction through specific recognition and binding to the phosphorylated 5′-end of the double-stranded DNA. λ-Exo also shows some cleavage activity towards single-stranded DNA, but with a much lower activity level than that towards double-stranded DNA. 7 Furthermore, the λ-Exo recognition site can be directly added to the double-stranded DNA by modifying the PCR primers. It also shows high enzymatic cleavage activity, with enzymatic cleavage activity per unit showing a value 10-fold that of exonuclease III. Based on these results, λ-Exo is a suitable enzymatic tool for our detection system.

Optimization of the Concentration of λ-Exo
The efficiency of λ-Exo in the enzymatic digestion of double-stranded DNA is high.
Theoretically, one unit of λ-Exo can release 12 mononucleotides per second. However, its cleavage efficiency varies greatly among different DNA sequences. 14,15 Enzymatic of that in the target-free group. The electrical signals did not decrease significantly as the λ-Exo concentration increased. Similar to that in the target-free group, the decrease in electrical signals when the concentration of λ-Exo exceeded 0.3 U/μL may be attributed to nonspecific cleavage. Our results indicate that 0.2 U/μL of λ-Exo is the optimal concentration for HEC reaction.

Electrical Signals
The electrical signals of the C/R1/A and C/R2/A sandwich structures ( Figure 1) designed with different probes and locations of biotin were compared. The results ( Figure 6) showed that the hybridization product C/R2/A yielded the higher electrical signals than that of the conventional structure (C/R1/A) in which biotin is conjugated away from the electrodes. Previous studies showed that the electrochemical signal is affected by the distance between electroactive materials and the electrode surface. Fan et al. manipulated the distance between ferrocene and the electrode surface to detect DNA. 17 Xiao et al. examined the changes in electron transfer efficiency caused by changes in the distance between methylene blue molecules and electrode surface to detect hybridization. 18 In this study, the current was produced by electroactive materials, bisazobenzidine, which originate from the HRP-catalyzed oxidation of TMB by H2O2 following the binding of biotin to streptavidin-HRP. The electron transfer efficiency increased along with the distance between bisazobenzidine and gold electrode surface decreasing, and electrical signals increased subsequently.

Detection of Synthetic Segment of Wild-type EGFR Gene
The reaction was carried out under optimal conditions with different concentrations of target DNA, i.e., wild-type EGFR DNA (0.01-1 nM) and the ratio of target to auxiliary DNA ranging from 1:100 to 1:1. The results showed that the electrical signals decreased significantly as the concentration of target DNA increased and the ratio of target to auxiliary DNA decreased. The decrease in electrical signals was particularly significant when the concentration of target DNA was <0.1 nM and the ratio of target to auxiliary DNA was >1:10. A greater number of HEC cycles in the reaction solution led to a lower amount of residual auxiliary DNA and thus a lower electrical signals.
However, the decreasing trend in electrical signals was decelerated by an increase in the concentration of target DNA, because the reduction of the ratio of target to auxiliary DNA limited the number of HEC cycles and prevented further reduction of electrical signals. Additionally, the presence of target DNA at relatively higher concentrations may influence the detection of residual auxiliary DNA. Therefore, the electrochemistry biosensor in this study is advantageous for detecting target DNA at low concentrations.
The detection limit (defined as S/N=3) was 10 pM, which was lower than our previous methods. Additionally, the concentration ratio of target to auxiliary DNA of >1:10 is conducive to the reaction.

Distinguishing Wild-type DNA from Deletion Mutants of EGFR Using Biosensors
To evaluate the specificity of this biosensor, wild-type EGFR DNA (which is fully complementary to the auxiliary DNA) and mutant EGFR DNA with three different types of deletion mutations were assayed under the same conditions. The target DNA, including wild-type DNA, del1, del3, and del5, were detected at a final concentration of 0.1 nM each with the ratio of 1:10 to auxiliary DNA, respectively. In the absence of λ-Exo, there was no significant difference between wild-type and mutant DNAs.
Whereas in the presence of λ-Exo, the electrical signals of the wild-type group were significantly lower than that of the deletion mutant groups (equivalent to only 26.7% of the deletion mutant groups), which showed high specificity of our biosensor. The complete complementarily hybridization between the wild-type EGFR DNA and auxiliary DNA favored the process of HEC cycles and thus significantly reduced the electrical signals. In contrast, hybridization between the deletion mutant and auxiliary DNA was rather weak because of the imperfect complementarity. Furthermore, even partial hybridization between the deletion mutant and auxiliary DNA was formed, the absence of double strands at the phosphorylated 5′-end due to the deletion mutation locating in 5′-end, significantly lower the recognition and enzymatic cleavage ability of λ-Exo as well as efficiency of HEC cycles.
This study combined "BSA-based probe carrier electrochemical DNA biosensors" with an exonuclease-assisted HEC signal amplification system to construct a complex system with an amplifying effect achieved by enzymatic digestion in a liquid homogeneous medium. Hybridization between target and auxiliary DNA into a double-stranded structure initiated the recognition and enzymatic digestion of the auxiliary DNA by λ-Exo, resulting in HEC cycles that allowed signal amplification through repeated recycling of a small amount of target DNA. We also optimized other conditions, such as the HEC reaction time and λ-Exo concentration. Additionally, we examined the effect of the distance between the electroactive materials and electrode surface on the electrical signals. Our results showed that the decreased distance between the electroactive materials and electrode surface resulted in greater electrical signals, thereby amplifying the detection signal. The biosensor constructed in this study showed a lower detection limit (10 pM for wild-type EGFR DNA) than the system using bovine serum albumin alone. Our biosensor also showed excellent specificity and could distinguish between wild-type and deletion mutants of EGFR. In conclusion, we constructed a signal amplification detection system successfully, and enables detection and evaluate the status of trace EGFR DNA by using electrochemical DNA biosensors.
Compared with traditional methods, the proposed biosensor detection method exhibits advantages such as time-saving, economic, high selectivity et al. which will urge this assay to be a promising method for the potential use in medical diagnosis for lung cancer patients. Furthermore, these strategies are expected to have further extensive

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