Development of a competitive chemiluminescent assay for quantitative determination of TP53 fusion protein using reagent strips

doi:10.21203/rs.3.rs-2202870/v1

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Development of a competitive chemiluminescent assay for quantitative determination of TP53 fusion protein using reagent strips

https://doi.org/10.21203/rs.3.rs-2202870/v1

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published 14 Feb, 2024

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Recent studies have shown that almost half of all cancers occur due to DNA damage. For the early diagnosis of cancer, a highly sensitized and swift identification for TP53 is needed since the corresponding TP53 protein is effectively recognized as “the guardian of the genome.” To improve the detection sensitivity, numerous analytical methods were previously used for the determination of the TP53 protein, including denaturing high-performance liquid chromatography and enzyme-linked immunosorbent assay (ELISA). Currently, immunochromatographic tests (ICTS) that are simple to use, stable over time, and show low interference are regarded as valuable tools for the quick screening of food and environmental monitoring along with clinical diagnosis. ICTS often have limited sensitivity even if a variety of novel reporters possessing optimum photostability and improved brightness are used as the signal-intensity reporters. Compared with N-(4-aminobutyl)-N-(ethylisoluminol) or luminol, a novel luminescent probe, 2',6'-diMethyl-4'-(N-succiniMidyloxycarbonyl) phenyl-10-sulfopropylacridiniuM-9-carboxylate (NSP-DMAE-NHS) has achieved a much higher efficiency, improvement in the biosensor's performance, and amplification of the signal without causing any damage to the biomolecule in terms of its biochemical activity. In this study, the reagent strip method was initially used to detect TP53 fusion protein by combining the advantages of NSP-DMAE-NHS and immunochromatography. In our experiment, the control and study lines on the strips were immobilized through HRP-conjugated goat anti-rabbit IgG and TP53 antigen, respectively. The optimized concentration of the anti-TP53 antibody-NSP-DMAE-NHS immunoconjugates was then added to the TP53 antigen samples. After, the test strips were inserted and left in the aforementioned buffer solution for an additional 20 minutes. Finally, a lab made luminous measurement device was used to analyze the corresponding control and study lines on the strips. Under optimized conditions, this method was found to be ultrasensitive, with a wide range of linear responses from 0.0008 ng mL^-1 to 1 µg mL^-1and a limit of detection of 0.0008 ng mL^-1 (0.013 pM). Thus, a novel competitive chemiluminescent assay based on reagent strips was established for the determination of the TP53 fusion proteins. The strategy has potential applications for ultrasensitive detection in the early diagnosis of cancer.

chemiluminescent quantitative determination

reagent strips

NSP-DMAE-NHS

TP53 antigen

In our earlier work, a quick and easy-to-use competitive chemiluminescence DNA assessment using Au-MNPs was constructed for detecting genomic p53 [1]. However, this strategy of using modified magnetic nanoparticles as carriers for p53 gene detection still has some limitations. The TP53 protein, also referred to as "the guardian of the genome," is crucial for the processes that repair a damaged genome [2, 3]. According to the theory, it acts as a tumor suppressor and activates downstream genes that impede growth and/or invasion upon binding to a TP53-binding site. TP53 is also the most frequently altered gene in human malignancies under conditions of cell stress, such as DNA damage caused by UV rays. Mutants of TP53 are widespread in numerous human cancers and unable to bind to the consensus DNA-binding site, which results in the loss of tumor suppressor action [4, 5]. Approximately 50% of all human cancers are recognized as an outcome of DNA damage. A variety of cellular stresses regulate various target genes that promote apoptosis, cell cycle arrest, senescence, DNA repair, or changes in metabolism [6, 7]. TP53 protein has therefore been previously determined using various analytical methods, including the use of denaturing high-performance liquid chromatography [8], the polydimethylsiloxane-assisted bead matrix [9], portable surface plasmon resonance biosensors [10, 11], biosensors based on field-effect transistors [12, 13], surface-enhanced Raman scattering spectroscopy [14], and electrochemical biosensor technology [15, 16], with enzyme-linked immunosorbent assays (ELISAs) [17–19]. However, each such technique requires the pretreatment of samples by skilled laboratory personnel owing to the complicated electrode materials and expensive equipment. Additionally, they are time-consuming, and high sensitivity is difficult to attain. Currently, immunochromatographic tests (ICTS) are regarded as a valuable tool for quick screening of food and environmental monitoring along with clinical diagnosis when compared to conventional approaches [20]. ICTS are rapid tests that are simple to use, stable over time, and show low interference [21–23]; therefore, immunochromatographic test strips combined with portable detectors are being manufactured by various manufacturers. Traditional ICTS often have limited sensitivity due to insufficient brightness when utilizing gold nanospheres as signal-intensity reporters [24]. Subsequently, ICTS applications designed for the detection of ultralow concentrations of analytes have now used a variety of novel reporters possessing optimum photostability and improved brightness; these include carbon nanoparticles [25], Fe₃O₄ nanoparticle aggregates [26], luminescent quantum dot beads [27], and upconverting phosphors [28]. Due to the ease of its conjugation to antibodies, its stability and its intense color, gold in its colloidal form has been the most frequently used immunochromatographic label [29, 30]. Unfortunately, gold cannot quantitatively determine the analyte, and flocculation and aggregation of gold in solution are unsuitable for large-scale ICTS production [31].

The probe of the analyte also impacts the analytical performance and is an important factor for immunoassays of detectable signals. Currently, chemiluminescence (CL) reactions are catalyzed using N-(4-aminobutyl)-N-(ethylisoluminol) or luminol as a luminescence reagent [32–37]. As a novel luminescent probe with high efficiency, NSP-DMAE-NHS demonstrates a much higher efficiency [38], improved selectivity, and enhanced emission intensity for quantitative analysis [39]. NSP-DMAE-NHS exhibits outstanding potential for the immobilization of biomolecules (proteins, DNA) with a carboxyl group, improves the biosensor's performance, and amplifies the signal without causing any damage to the biomolecule in terms of its biochemical activity.

In our previous work, modified magnetic nanoparticles were utilized as carriers to carry out specific binding of gene fragments; these fragments used the magnetic properties of the magnetic particles to carry out the separation operations. Specific binding occurred in the solution phase, and the solution to be tested was not easily preserved; thus, it could not be tailored into a kit for wide-scale utilization in health care. Therefore, based on the results of our earlier investigation, we focused on the applications of chemiluminescence technology and immunochromatographic methods. A novel competitive chemiluminescent assay for the determination of the TP53 fusion protein based on reagent strips was established, with NSP-DMAE-NHS as an efficient luminescence reagent. In this study, the TP53 antigen together with HRP-conjugated goat anti-rabbit IgG were immobilized on control and study lines on the strip, respectively. Then, the (anti-TP53)-(NSP-DMAE-NHS) immunoconjugates were prepared and detected using different concentrations of the TP53 antigen samples with the optimal concentrations. After being immobilized on the control and test lines of the strips, the TP53-(anti-TP53)-(NSP-DMAE-NHS) immunoconjugates and the remaining (anti-TP53)-(NSP-DMAE-NHS) conjugates were determined using a luminous analytical platform. This method combined portable equipment with express testing, enabling the results to be recorded, followed by an immediate transfer of the results with a variety of current telecommunications methods. Thus, the immunochromatographic systems was more competitive in carrying out nonlaboratory analyses. The principle of the chemiluminescent quantitative determination of the TP53 protein based on the reagent strips is shown in Fig. 1. Compared with previous work, our detection method focused on an immunoassay and was characterized by its simplicity, efficiency, high sensitivity, and fast speed. This strategy used reagent strips as the carrier for specific immunobinders and has provided a solid foundation for the preparation of commercial kits and their application in community hospitals and other healthcare institutions.

Materials and Reagents

The TP53 antigen, anti-TP53 antibody, together with HRP-conjugated goat anti-rabbit IgG were procured through Sangon Biotechology Inc. (China). The TP53 antigen was a TP53 fusion protein corresponding to region-derived amino acids of human tumor protein TP53, and the anti-TP53 antibody was an anti-TP53 rabbit polyclonal antibody immunogen of the fusion protein of human TP53. Sephadex G-25 and L-lysine were procured from Seebio (Shanghai, China). Bovine serum albumin (BSA) and 2-(N-morpholino)-ethanesulfonic acid (MES) were obtained from J&K Scientific, Ltd. 2',6'-DiMethyl-4'-(N-succiniMidyloxycarbonyl) phenyl-10-sulfopropylacridiniuM-9-carboxylate (NSP-DMAENHS) was procured from Helison Biotechnology Co., Ltd. (Xiamen, China). N,N-Dimethylformamide (DMF), 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide hydrochloride, N-hydroxysuccinimide (NHS), and H₂O₂ were acquired from Sigma, China. Nitric acid cellulose film (Sartorius CN140), PVC substrate (DB-6), glass cellulose film (Ahlstrom 8964), and absorbent paper (H5072) were purchased from Shanghai Jieyi Biotechnology Co., Ltd. All other chemicals were of analytical grade and procured through Tianjin Tianli Chemical Regents, Ltd. A peristaltic pump (TYD01) was purchased from Lead Fluid Technology Co., Ltd. All experiments utilized deionized water, and the chemicals were used without any further purification or treatment.

Luminescent analytical platform development

The self-designed luminescent analytical platform is shown in Fig. 2; the platform consisted of a photomultiplier (PMT) tube, a peristaltic pump, a rotating platform, a dark box, and a photon counting unit (H8259-01, Hamamatsu). The photomultiplier tube functioned as a photoelectricity-detecting apparatus and collected the light intensity represented by the number of photons per unit of time. The automatic control of the rotating platform was placed into the black box to prevent outside light. The photomultiplier tube was placed straight above the rotating platform corresponding to the reagent strips. Thus, when NSP-DMAE-NHS was stimulated to release 430 nm wavelength photons on the reagent strip, its luminescence was effectively collected. A peristaltic pump was used to inject the trigger solution onto the control lines and test lines of the strips. Moreover, the chemiluminescent emission intensity derived through the analyte was measured; the emission signal was transferred, observed and manipulated in real time using a computer.

Immobilization of the TP53 antigen on the reagent strips

The reagent strips were prepared using nitric acid cellulose film, PVC substrate, glass cellulose film, and absorbent paper. The entire assembled plate was cut into reagent strips of identical size by a cutting machine. The length and width for individual reagent strips were 60 and 2.5 mm, respectively. Strips were kept in a dry and sealed environment at normal temperature. Analytical performance is also markedly influenced by matrix effects (MEs). Generally, two strategies to reduce the MEs are (1) using the most effective extraction process and (2) diluting the test sample prior to measurement. Presoaking the starting pad of the test strip with proteins can greatly diminish the MEs since the pad of the test strip can potentially adsorb a variety of interfering substances.

The working principle of the reagent strip is to capture specific antigens through specific antibodies. The preassembled reagent strips were marked for the positions of test lines and control lines. The TP53 antigen was prepared at a specific concentration along with an HRP-conjugated goat anti-rabbit IgG solution. The HRP-conjugated goat anti-rabbit IgG and TP53 antigen were immobilized on the control and study lines of the strips, respectively. The reagent strips were then placed in a vacuum drying oven for 30 minutes at 37°C.

Underlying principle for the competitive chemiluminescence production and detection of TP53

Figure 1 depicts the reaction process. On the control and study lines of the strips, the TP53 antigen and HRP-conjugated goat anti-rabbit IgG were immobilized, respectively. The produced reagent strips were submerged in 5% BSA solution for 20 minutes, followed by 20 minutes in PBS buffer solution. To create the standard samples, the TP53 antigen was dissolved in buffer solution at concentrations of 0.0005, 0.001, 0.01, 0.1, 1, 10, 100, and 1000 ng mL^-1. The optimum quantity of the (anti-TP53)-(NSP-DMAE-NHS) conjugates was added to the aforementioned solutions, and they were then incubated at 37 ℃ for 30 minutes. Subsequently, the reagent strips were placed into the above solution for another 30 minutes. The presence of liquid within the carrier resulted in the partial immobilization of compounds on the reagent strips. The reagent strips were washed with the PBS buffer solution. Finally, the corresponding control and study lines on the strips were analyzed through an in-house luminescent measurement system. Here, NaOH and HNO₃ + H₂O₂ functioned as the trigger and pre-trigger, respectively. Then, a 1 µL pre-trigger was initially added dropwise onto the control and study lines upon strips, and 3 µL of the trigger was injected onto the control and study lines strips, respectively, through a peristaltic pump. Moreover, the photomultiplier tube, which functioned as a photoelectricity-detecting apparatus, collected light intensity by measuring the photon number within a unit time interval. Chemiluminescence emission intensity was quantified and converted into an electrical signal.

Preparation of (anti-TP53)-(NSP-DAME-NHS) conjugates

The (anti-TP53)-(NSP-DAME-NHS) conjugates were produced with some modifications to the previous strategies [40]. The anti-TP53 antibody concentration was 0.8 mg mL^-1 (0.05% NaN₃, 40% glycerol, pH 7.4 PBS). NSP-DAME-NHS was used to label 100 µL of purified anti-TP53 antibody; NSP-DAME-NHS (1 mg) was solubilized in dry DMF (316 µL) (Fig. S1). The reaction solution was shaken for an hour at room temperature to complete the reaction before being quenched for a further 15 minutes with 100 microliters of the L-lysine solution (10 mg dissolved per milliliter of sterile distilled water). Gel filtration was used to separate the labeled anti-TP53 antibody conjugates from the unbound NSP-DAME-NHS using a 25 cm column (Sephadex G-25) that had been equilibrated with 0.1 M PBS at pH 7.4. CPS is the counts per second. The anti-TP53 antibody was labeled with NSP-DMAE-NHS, as shown by the peak at 11 mL. The conjugates featured a significant chemiluminescence peak and a strong absorption peak at 260 nm. The peak at 40 mL showed that the chemiluminescence peak was only caused by the additional NSP-DMAE-NHS. The results are presented in Fig. 3. Evidently, the anti-TP53 antibody labeled with NSP-DMAE-NHS was eluted at approximately 11 mL. It was gathered, enriched, and kept at 4°C in single-dose vials.

To assess the sensitivity, the aforementioned (anti-TP53)-(NSP-DAME-NHS) conjugate solution was consecutively diluted in purification buffer from 1:10 to 1:10^− 5. Then, our self-designed luminescent measurement equipment was used to measure the diluted (anti-TP53)-(NSP-DMAE-NHS) conjugates. A peristaltic pump was employed to inject the trigger solution into individual well tubes every 2 seconds. The number of photons in a unit of time was used to determine light intensity collected by the photomultiplier tube as a photoelectricity-detecting device. The computers continuously monitored and processed the signals. The moles of the anti-TP53 antibody were plotted vs. the relative light unit (RLU) that was emitted, as shown in Fig. 4(a). The variation in chemiluminescent intensity over time is depicted in Fig. 4(b). It was determined that the sensitivity was at least 1.8x10^-17 mol.

Optimization of the quantity of NSP-DMAE-NHS-labeled anti-TP53 antibody

Prior to developing standard curves across the TP53 antigen dose range, the exact quantity of NSP-DMAE-NHS-labeled anti-TP53 antibody was optimized. Various amounts (1 µL, 5 µL, 10 µL, 20 µL, 35 µL, 40 µL, 50 µL, 60 µL) of (anti-TP53)-(NSP-DMAE-NHS) conjugates were added to the tubes. Then, the reagent strips fixed with the antigen were placed into the reaction tube, and specific immune binding occurred as the fluid was moved. The intensity of chemiluminescence for various concentrations of (anti-TP53)-(NSP-DMAE-NHS) conjugates is shown in Fig. 5. (Anti-TP53)-(NSP-DMAE-NHS) was present at a concentration of 0.01 mg mL^− 1. Evidently, the intensity of the chemiluminescence linearly increased with respect to the dosage for (anti-TP53)-(NSP-DMAE-NHS) within the 1–50 µL range, and thus, saturation was achieved at 50 µL. The steric effect was presumably responsible for this outcome [41]. Specifically, the (anti-TP53)-(NSP-DMAE-NHS) and TP53 antigen reaction on the test line of strips could be affected by the steric effect. Once there were more than 50 µL (anti-TP53)-(NSP-DMAE-NHS) conjugates, they interacted with each another and underwent structural changes that caused difficulty for the antibodies to detect the antigen. For the purpose of achieving high sensitivity, an optimal volume of 30 µL of the (anti-TP53)-(NSP-DMAE-NHS) conjugates was designated for the competitive reaction; this aligned with the concentration causing 50% inhibition (IC₅₀).

Calibration curve for the TP53 antigen analytes

The mixing of various concentrations of the TP53 antigen (0.0005, 0.001, 0.01, 0.1, 1, 10, 100, and 1000 ng mL^− 1) was carried out with a 30 µL solution of (anti-TP53)-(NSP-DMAE-NHS); this was followed by allowing the mixture to incubate at 37°C for 30 min. Subsequently, the reagent strips were added to the above buffer solution for another 30 minutes. The reagent strips were thoroughly cleaned twice using PBS with 0.1% Tween 20 after the reaction was finished, and the control and study lines upon strips were quantified using a lab made luminous measurement device. As shown in Fig. 6(a), chemiluminescence intensities linearly declined as the TP53 antigen concentrations increased. This was caused by the analyte and tracer's competitive immunological binding to the antibodies immobilized on the outermost surface. The calibration curve for the TP53 antigen dose and photon count peak is given in Fig. 6(b). The linear range of the TP53 concentration was from 0.001 ng mL^− 1 to 100 ng mL^− 1, and the regression equation was y = 27903.5-6770.5x (R² = 0.984), where x is the amount of TP53 antigen present and y refers to the number of photons. The limit of detection (LOD) was calculated using 3σ (where σ is the relative standard deviation of a blank control group, n = 6) and determined to be 0.0008 ng mL^− 1 [42]. The IC₅₀ value was approximately 0.24 ng mL^− 1. Figure 6(c) shows the chemiluminescence intensities of the control lines of the strips, which correspond to the different concentrations of the TP53 antigen. The chemiluminescence intensities linearly increased as the TP53 antigen concentrations increased. This result further clarifies why TP53-(anti-TP53)-(NSP-DMAE-NHS) conjugates were immobilized more on the control lines of the strips and less on the test lines of the strips under higher quantities of TP53 antigen in the sample. A detailed comparison between our method and other common detection techniques is provided in Table 1. The results were similar or superior to those of previously reported TP53 tests [43–45], and our method was found to have increased sensitivity in a wider linear range than the formerly employed methods [46, 47]. The higher binding ratio for the conjugates (anti-TP53)-(NSP-DMAE-NHS) binding TP53 antigen on the test line of the strips together with the high emission efficiency of NSP-DMAE-NHS could have significantly contributed to this result. In our previous work, modified magnetic nanoparticles were used as carriers to detect the p53 gene, and the method had elevated sensitivity, a wide linear detection range, and the characteristics of being noncomplex and fast. However, the assay needed to be performed in the laboratory and required professional personnel to conduct the process of experimental testing, and the operation steps were relatively professional, especially during magnetic separation. In addition, specific binding occurred in the solution, and the solution to be tested was not easily preserved. By comparison, this method focused on an immunoassay that was simple, efficient, and fast. There was no need for expensive equipment or professional technicians in the experimental operation, and the prepared reagent strips were more easily preserved. Although the sensitivity and linear range did not improve, this strategy of using reagent strips as the carrier for specific immunobinding has facilitated a certain foundation for the preparation of commercial kits and their application in community hospitals and other healthcare institutions.

Table 1

Comparison of this method and other typical methods
DNA assay method	Detection limit	measurement range	References
non-competitive electrochemical nanofiber biosensor	50 fM	——	[42]
non-competitive electrochemical peptide nucleic acid (PNA) probe biosensor	0.682 nM.	——	[43]
Competitive luminol DNA assay	3.8 pM	0.01–0.5 nM.	[44]
sequence-specific molecularly imprinted PoPD electrode	——	0.01-300fM	[45]
non-competitive electrochemical DNA Sensor	0.8 nM	1.0-1000 nM	[46]
non-competitive electrochemiluminescence	22.8 fM	0.2–200 pM	[47]
Competitive improved CL DNA assay	0.16pM	0.001–6600 ng mL^− 1	Our previous work [1]
competitive chemiluminescent quantitative determination of TP53 fusion protein base on reagent strips	0.13pM	0.0008-1000 ng mL^− 1	This work

Selectivity and reproducibility of the chemiluminescence analysis (CLIA) detection of the TP53 fusion protein

Our proposed immunosensor's selectivity was examined as the last stage in the assessment of its effectiveness by hybridizing with (anti-TP53)-(NSP-DMAE-NHS) conjugates linked to the TP53 antigen, using the reagent strips as a carrier. For this purpose, the three tubes with the same concentrations (0.1 ng mL^− 1) of TP53 antigen were labeled as 1, 2, and 3 for convenience. The concentrations of the fourth and fifth tubes were 1 and 10 ng mL^− 1, respectively. The detection systems of 1 and 2 were separately incubated with different kinds of proteins, including CA125 (0.1 ng mL^− 1) and alpha-fetoprotein (0.1 ng mL^− 1), and the detection system of 3 was replaced with a blank buffer solution [48]. Moreover, a 30 µL solution of (anti-TP53)-(NSP-DMAE-NHS) was mixed with the above-incubated solution for 30 minutes before following the previous steps. The chemiluminescence signals of each protein-produced reagent strip were subsequently measured. The photon count peaks remained unaffected as a result of the inclusion and interference of unrelated proteins, according to the data gathered and shown in Fig. 7. The chemiluminescence intensities of the TP53 and mixed samples were essentially consistent, and the sensitivity of the chemiluminescence immunosensor to the other interferences was mild and inconsequential. According to the findings, the TP53 antigen could be easily distinguished from CA125 and alpha-fetoprotein (AFP) using the (anti-TP53)-(NSP-DMAE-NHS) conjugates. The immunosensor demonstrated excellent dependability regardless of the presence of other proteins or interferences and showed high sensitivity and good selectivity toward the TP53 protein. Thus, it functioned as a unique detection platform for cancer diagnosis at early stages [49–50]. Our proposed assay's reproducibility was tested thrice via measurements of the designed competitive chemiluminescent assay in the presence of 0.001 ng mL^− 1 TP53 antigen. The relative standard deviation (RSD) was 5.3%, demonstrating the method's high level of reproducibility. Additionally, the competitive chemiluminescent assay's stability was examined after one week of storage at 4°C, and 97.1% of the initial sensitivity was retained.

A new competitive chemiluminescent assay based on reagent strips was developed to identify the TP53 fusion protein. In our proposed method, for the first time, the benefits of immunochromatography and NSP-DMAE-NHS were combined for the detection of the TP53 fusion protein. Additionally, our method had a detection limit of 0.0008 ng mL^-1 (0.013 pM); thus, our approach had strong selectivity and high sensitivity for the detection of the TP53 fusion protein. Our design significantly minimized the amount of reagent needed for the test, which could greatly reduce the cost per assay. If this innovative biosensor platform becomes commercially viable, it could be extended for cancer diagnosis, early cancer detection, and the detection of other biomarkers.

Acknowledgements The authors would like to thank the (1) Science and Technology Project of Hebei Education Department (No. BJ2020209) and (2) Technology Research and Developmental Program in Science and Technology of Handan City (No. 19422111001-10) for their support in performing this research work.

Author Contribution Linyu Wang: Development of experimental analysis and manuscript writing. Zhifang He: chemiluminescent method and analysis. Jianye Li: Preparation of reagent strips.

Funding This work was supported by the Science and Technology Project of Hebei Education Department (No. BJ2020209) and the Technology Research and Developmental Program in Science and Technology of Handan City (No. 19422111001-10).

Conflict of interest The authors declare that they have no competing interests.

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published 14 Feb, 2024

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Editorial decision: Accept without change
04 Feb, 2024
Editor invited by journal
06 Nov, 2023
First submitted to journal
03 Nov, 2023

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Development of a competitive chemiluminescent assay for quantitative determination of TP53 fusion protein using reagent strips

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Experimental

Materials and Reagents

Luminescent analytical platform development

Immobilization of the TP53 antigen on the reagent strips

Underlying principle for the competitive chemiluminescence production and detection of TP53

Results and Discussion

Preparation of (anti-TP53)-(NSP-DAME-NHS) conjugates

Optimization of the quantity of NSP-DMAE-NHS-labeled anti-TP53 antibody

Calibration curve for the TP53 antigen analytes

Selectivity and reproducibility of the chemiluminescence analysis (CLIA) detection of the TP53 fusion protein

Conclusions

Declarations

References

Status:

Journal Publication

Version 1