Re-engineering of peptides with high binding affinity to develop an advanced electrochemical sensor for colon cancer diagnosis
Graphical abstract
Introduction
Colorectal cancer (CRC), a malignant tumor found in the large intestine or rectum, is one of the most common cancers and the fourth leading cause of cancer-related death [[1], [2], [3], [4]]. In recent times, there has been a significant reduction in the survival and treatment success rates of CRC [5,6] owing to early diagnosis. A statistical analysis revealed that adenocarcinoma was found to constitute 95% of all CRCs and that the transition of adenoma to carcinoma is the most critical process in CRC development [7]. CRC could also be effectively controlled and curable if the premalignant lesion (adenoma) is detected and removed before invasion. Currently, several clinical diagnostic methods are used for CRC. Among these methods, colonoscopy is considered the gold standard for early CRC diagnosis [[8], [9], [10]]. However, a substantial proportion of patients may find colonoscopy inconvenient due to the high medical cost and the uncomfortable procedure [[11], [12], [13]]. Therefore, because of increasing demands for the development of a cost-effective and reliable diagnostic method, alternatives such as DNA analysis, determination of carcinoembryonic antigen levels, and antibody-based assays have been suggested [[13], [14], [15], [16], [17]]. In particular, these methods can distinguish patients with CRC from healthy controls but cannot distinguish precancerous adenoma and carcinoma, which is crucial for early CRC diagnosis. Further, although these methods seem simpler and more affordable, a more sensitive diagnostic technology needs to be developed to distinguish precancerous adenoma and carcinoma.
Leucine-rich α-2-glycoprotein-1 (LRG1) plays a major role in apoptosis and cell survival in patients with CRC [1]. In particular, previous studies have confirmed that LRG1 expression levels in the plasma from patients with carcinoma were more than 2-fold higher than those in the plasma samples of patients with adenoma [1]. These results suggest that LRG1 is related to the transition from adenoma to carcinoma. Currently, an antibody-based detection assay is the most widely used method for detecting LRG1. Antibodies serve as important capture reagents in several fields such as the clinical or pharmaceutical analysis, diagnosis, and treatment of various diseases. However, they have limitations such as being relatively large in size and having cross-reactivity in immunoassay tests. Additionally, because most antibodies are produced in mammalian cells, they can require relatively long times for mass production, and are expensive. In contrast, affinity peptides have a small size and simple structure and are relatively stable in a variety of environmental conditions. Therefore, affinity peptides are gaining increasing attention over conventional affinity reagents in many fields [18,19].
Phage display is one of the most optimized assays for screening and/or identifying high affinity peptides for wide use in many applications such as diagnosis, small molecule-based drug development, drug delivery, and pharmaceutical analysis [16,[20], [21], [22], [23], [24], [25]]. Newly screened peptides or proteins are used for protein-protein or protein-peptide interactions and for identification of sequences specific to their target for replacing affinity reagents that specifically bind to target proteins or metals [26,27]. Researchers have developed a peptide-based sensor capable of recognizing and capturing norovirus [28], procalcitonin (PCT) [27], and neutrophil gelatinase-associated lipocalin [29] using a combination of phage display technology and electrochemical analysis. For example, electrochemical-based biosensors have been widely applied in many fields because they have high sensitivity, are easy to control, and allow for label-free detection and miniaturization in a point-of-care setting [[30], [31], [32]]. Furthermore, the working electrode surface of an electrochemical sensor can be composed of many materials such as gold, silver, and graphene, and the immobilization of target-specific probes such as DNA, antibody, and protein on the working electrode surface can enable targeted specific detection in electrochemical analysis. Because of these characteristics, electrochemical biosensors stand out for extensive development toward point-of-care diagnostics [13,[33], [34], [35]]. Among electrochemical sensing methods, square wave voltammetry (SWV)-based detection has been widely used for the quantitative detection of various compounds such as drugs, biomolecules, and environmental pollutants. SWV has advantages such as short analysis times, simplicity, high sensitivity, and cost-effectiveness compared to other modes of analysis.
In a previous study, we screened short linear affinity peptides specific for LRG1 protein using phage display technology and evaluated their binding affinities by electrochemical analysis [22]. We then selected the best affinity peptides containing positive charges (His) or negative charges (Asp) and confirmed that they can be used to detect their target proteins in actual patient samples. Despite these successful results, the direct immobilization of short peptides on a gold electrode can sometimes lead to instability as it does not control the densities per gold surface area, and thus more optimized sensors are needed for real testing.
Considering these findings, in this study, we used a rational design approach with in silico modeling and synthesized advanced affinity peptides for an electrochemical sensor. In addition, a new linker was used to specifically immobilize the affinity peptide on a gold electrode with a surface chemistry approach. Finally, we tested the performance of this advanced sensor system in distinguishing patients with adenoma from those with carcinoma.
Section snippets
Chemicals
Recombinant full length human leucine-rich alpha-2-glycoprotein1 (LRG1) was purchased from Origene (TP306780, Rockville, MD, USA). KNO3, 1,6-hexanedithiol (HDT) 6-mercaptohexanol (MCH), benzoquinone, and human plasma were purchased from Sigma-Aldrich (St. Louis, MO, USA). All amino acid-substituted synthetic peptides (LRG1 BP3-1 to LRG1 BP3-8) were chemically synthesized (>95% purity) by Peptron (Daejeon, Korea) (Table S1). The commercially available LRG1 ELISA detection kit was purchased from
Re-design and synthesis of affinity peptides for advanced characteristics
As mentioned earlier, we have reported the development of an affinity peptide specifically binding to LRG1 protein, a biomarker of colon cancer [3]. In that study, the affinity peptide specific to LRG1 protein had the sequence QDIMDLPDINTL, with a Cys residue at the C-terminus for binding the gold surface and a flexible linker (-GGGGS-) for flexibility. This suggested a possibility for the diagnosis of colon cancer, however, there were some hurdles to be crossed. First, the density of peptide
Conclusion
In CRC, it is crucial to distinguish between adenoma and carcinoma. Therefore, in this study, we designed improved affinity peptides for an advanced electrochemical sensor for identifying the adenoma to carcinoma transition, which would be useful in point-of-care testing. To increase the sensitivity, stability, and reproducibility of the sensor, eight peptides with different linkers were rationally designed by in silico modeling and were chemically synthesized, and a new specific immobilization
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF-2017R1A2A2A05001037, NRF-2019R1A2C2084065).
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