Modification-free amperometric biosensor for the detection of wild-type p53 protein based on the in situ formation of silver nanoparticle networks for signal amplification

https://doi.org/10.1016/j.ijbiomac.2020.04.271Get rights and content

Abstract

Sensitive and accurate quantification of wild-type p53 protein is of great importance for biological research and clinical diagnosis. Herein, a modification-free amperometric biosensor was proposed for sensitive detection of wild-type p53 protein by the signal amplification of silver nanoparticles (AgNPs) networks formed in situ on electrode surface. Double-stranded DNA (dsDNA) probe containing two consensus sites was immobilized on gold electrode surface to capture wild-type p53 protein. The cysteine thiol and amine groups on the exterior of the protein allowed for the attachment of bare AgNPs through the Agsingle bondS or Agsingle bondN interactions. Meanwhile, benzene-1,4-dithiol (BDT) molecules in solution triggered the assembly of more AgNPs on electrode surface through the Agsingle bondS interactions, thus leading to the in situ formation of AgNPs networks for signal amplification. The target at the concentration as low as 0.1 pM can be readily determined. This method was further applied to determine wild-type p53 protein in spiked human serum and cell lysates with satisfactory results. Moreover, the biosensor is regenerative and does not require the modification of AgNPs with recognition element for signal readout. The modification-free strategy can potentially be applied to develop novel biosensors for detection of other biological macromolecules.

Introduction

p53 protein, a tumor suppressor and transcription factor encoded by TP53 gene, plays important roles in multiple central cellular processes (e.g. DNA repair, cell cycle control and apoptosis) [1]. Concentration of wild-type p53 protein in normal cells is usually ultralow [2,3]. Mutation or inactivation of p53 gene in more than 50% of human cancers can cause the decrease of wild-type p53 protein and the accumulation of mutated p53 protein [4]. Following the death and destruction of tumor cells, p53 protein is released from cancer cells and circulated in the blood of cancer patients. Serum levels of p53 can accurately indicate tissue alteration at the gene and/or protein level [5]. Therefore, it is of great significance to achieve sensitive detection of wild-type p53 protein at ultralow concentration in human serum for clinical research and early diagnosis of cancers.

The classic methods for detection of p53 protein, such as enzyme-linked immunosorbent assay (ELISA) [6], electrophoretic mobility shift assay [7], immunostaining [8] and immunohistochemistry [9], generally involve in semi-quantification, low sensitivity and tedious experiment steps. In order to meet the increasing demand for sensitive detection of p53 protein, researchers have focused considerable efforts to establish novel analytical methods, including electrochemistry [[10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25]], electrochemiluminescence [26], photoelectrochemistry [27], localized surface plasmon resonance (LSPR) [28], surface enhanced Raman scattering (SERS) [29,30] [31], and immunochromatographic test strip [3]. Among them, electrochemical biosensors have attracted considerable attention because of their intrinsic advantages of simple operation, high sensitivity, speed response and low cost. However, the biosensors mainly based on the specific antibody-antigen interaction cannot distinguish the wild-type p53 and mutant p53. Human p53 protein composing of 393 amino acid residues is functionally divided into three distinct domains: the transactivation domain at the N-terminus, the central DNA-binding domain, and the oligomerization domain near the C-terminus. It has been revealed that four wild-type p53 molecules can assembly into a tetramer in solution by their C-termini. The resultant p53 tetramer can bind to sequence-specific sites in the consensus double-stranded DNA (dsDNA) but the mutant p53 protein loses the binding ability [32]. Several groups have investigated the dsDNA/p53 interaction and developed a few biosensors with the consensus dsDNA as the acceptor for selective detection of wild-type p53 protein [[33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43]]. Herein, we attempt to develop a simple and sensitive electrochemical biosensor for specific detection of wild-type p53 protein using the consensus dsDNA receptor.

To meet the urgent demand of ultrasensitive detection of biomarkers, electrochemical sensing strategies can be integrated with signal amplification to enhance the detection sensitivity. With the striking development of nanotechnology, various nanomaterials have been synthesized and used for designing of electrochemical biosensors with improving sensitivity, including noble metal nanoparticles (NPs), carbon nanostructures, metal-organic framework, metal oxide/sulfide NPs and other novel two dimensional (2D) nanomaterials [[44], [45], [46]]. The nanomaterials are usually employed as electrode modifiers to accelerate electron transfer and increase electrode surface area, as the carriers to load large numbers of enzyme labels or electroactive species, and/or as the reporters to amplify the electrochemical signal. For example, silver nanoparticles (AgNPs) modified with specific recognition element have been extensively utilized as the redox reporters to develop sensitive electrochemical biosensors for the analysis of cancer cells, DNA, microRNAs, proteins, drugs and metal ions because of their excellent electrochemical properties [[47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57]]. Wild-type p53 proteins in solution can form tetrameric oligomer. The tetramer can bind to dsDNA at the C-terminus with high binding affinity and each p53 monomer contains five surface accessible cysteine thiols on the exterior [38,58]. For this view, we developed an electrochemical biosensor for the detection of wild-type p53 protein based on the interaction of cysteine thiols and AgNPs. The signal was further amplified on the basis of benzene-1,4-dithiol (BDT)-induced in situ formation of AgNPs networks on electrode surface. The method has the advantages of high sensitivity and simplicity and does not require the modification of AgNPs nanolabels with recognition element for signal readout. Note that metal-free cysteine thiol and amine groups in most of proteins can attach onto the bare Au/Ag surface but dsDNA hybrids exhibit no interaction with bare Au/Ag nanoparticles [59]; the method may be extended to design novel biosensors by matching sequence-specific dsDNA acceptors.

Section snippets

Chemicals and reagents

Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), BDT, 6-mercapto-1-hexanol (MCH), tris-(hydroxymethyl)aminomethane hydrochloride (Tris–HCl), IgG, thrombin and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (Shanghai, China). p53 ELISA kits were provided by SenBeiJia Biological Technology Co., Ltd. (Nanjing, China). HPLC-purified oligonucleotides were ordered from Sangon Biotechn. Co., Ltd. (Shanghai, China). Their sequences were listed as follows: the thiolated single-strand

Detection principle of the method

The modification-free amperometric biosensor for detection of wild-type p53 protein was developed based on the high affinity of wild-type p53 protein toward consensus dsDNA. The detection principle is illustrated in Scheme 1. The mixed dsDNA/MCH self-assembled monolayers (SAMs) are performed on the surface of gold electrode as the bioreceptor to capture wild-type p53 protein and eliminate the non-specific adsorption. After the formation of the p53 protein/dsDNA complex, the five metal-free

Conclusion

In summary, we developed a modification-free electrochemical biosensor for sensitive detection of wild-type p53 protein. AgNPs networks formed in situ on the sensor electrode surface were utilized as the electroactive reporters for signal readout. The well-defined electrochemical signal originated from the solid-state Ag/AgCl reaction of AgNPs-based networks. The biosensor exhibited a wide linear range and a detectable concentration as low as 0.1 pM. In contrast to other nanomaterials-based

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

We acknowledge the financial support of the Henan Province University Innovation Talents Support Program (18HASTIT005) and the Science & Technology Foundation of Anyang City.

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