Colorimetric analysis of C-reactive protein via ‘jellyfish’ probe-based exonuclease III-assisted multiple-signal recycles

Postoperative infections are a significant complication that can increase organ dysfunction, hospital stays and mortality in patients receiving coronary artery bypass graft (CABG) surgery with cardiopulmonary bypass [1–3]. Infection rates ranging 12.9–30.8% have been seen in studies of this patient population [4]. Because of the inflammatory response that CABG causes, it is difficult to employ inflammatory markers to identify infections, leaving clinical assessment as the primary method for healthcare professionals. The significant prevalence of CABG surgery-related risk factors necessitates ongoing clinical surveillance for early detection, effective treatment and prevention. Increased levels of C-reactive protein (CRP), an acute-phase protein that is overexpressed in and after CABG, may be utilized as a tool to aid in the diagnosis, prevention and treatment of infection [5,6]. CRP is widely believed to be a superior indicator of inflammation. For a better understanding of CRP's roles in inflammation and an early assessment of CABG risks [7], sensitive monitoring of the expression level of CRP is in high demand.

Nowadays, common analytical tools such as ELISA, immunoturbidimetry and immunoluminometry can be used to perform conventional assays for the detection of CRP in clinical laboratories [8–10]. Although these technologies can achieve detection limits as low as 1 mg/ml, they are time-consuming and expensive. In addition, these methods require bulky equipment and skilled personnel for analysis. Because of these constraints, the ELISA technique cannot be used to diagnose acute-phase inflammation after CABG surgery or detect CRP in low-resource environments [11,12]. It is desirable in this situation to use alternative techniques for improving clinical diagnosis of acute-phase inflammation quantitatively by detecting and quantifying CRP in a quick, simple, label-free and cost-effective manner. In recent years, various methods have been proposed to measure CRP in point-of-care settings. For example, Ahn et al. developed a quantitative lateral flow assay test with a limit of detection (LOD) of 0.133 mg/ml CRP using the Alexa647 and a 1D fluorescence scanner approach [13]. In addition, Swanson et al. developed a CRP test method using near-infrared-LFAs that exhibited a dynamic range of 0.05–2.5 mg/ml [14]. However, these technologies still require expensive fluorescence analyzers capable of converting the fluorescence signal to an electrical signal for data processing. Thus, the creation of a portable platform for sensitive CRP detection in resource-constrained point-of-care conditions is necessary. Colorimetric assays can be used to quantify a specific analyte quantitatively by using straightforward portable optical detectors or color changes that are visible to the naked eye [15–17]. As a result, point-of-care CRP detection is very appealing for assessing the risk of acute-phase inflammation because early medication administration is essential [18,19]. However, colorimetric assays are commonly criticized for their low sensitivity, highlighting the urgent demand for a sensitive colorimetric approach for CRP detection.

Herein, a colorimetric assay for sensitive detection of low-abundance CRP in clinical samples by coupling exonuclease III (Exo III)-assisted multiple-signal recycles is proposed. In this method, an arch probe was designed to specifically bind with CRP and to initiate Exo III-based signal recycles. In the signal recycles, a ‘jellyfish’ probe is exploited to identify the initiator sequence released in the target recognition process and to mediate the triple-signal amplification via Exo III. The target CRP input signal can be amplified into a variety of DNAzyme outputs, creating green 2, 2′3- azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^-) for robust colorimetric and visual CRP detection. This device offers a straightforward, portable and sensitive platform for measuring CRP and has the potential to be used to evaluate inflammatory responses in patients receiving cardiopulmonary bypass and in clinical molecular diagnostics.

Materials & method

Material & reagents

The DNA oligonucleotides used in this research were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China). Details regarding the nucleic acids sequences employed for signal amplification are shown in Supplementary Table 1. Hemin, ABTS^2- and Exo III enzyme were purchased from New England Biolabs (Beijing, China). CRP was obtained from TaKaRa Biotech. Inc. (Dalian, China). Diethylpyrocarbonate, phosphate-buffered saline (PBS) solution, hydrogen peroxide (H₂O₂) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (MO, USA).

Construction of arch & ‘jellyfish’ probes

Arch probe

To assemble the arch probe, 5 μl aptamer sequence and 5 μl initiator sequence were mixed in a tube containing 15 μl PBS buffer solution. The mixture was incubated at 90°C for 10 min and cooled to room temperature (2°C /min). To test the feasibility of the arch probe, 10 μl arch probe was mixed with 0.1 μg/ml CRP (5 μl). The mixture was incubated at room temperature for 30 min.

‘jellyfish’ probe

A total of 5 μl linear hairpin probe was mixed in a tube containing 10 μl PBS buffer solution. The mixture was incubated at 90°C for 10 min and cooled to room temperature (2°C /min). Afterward, 5 μl of ssDNA chain was added to the mixture and the mixture was incubated at room temperature for 30 min.

CRP detection

A total of 5 μl arch probe was mixed with 5 μl CRP in a tube containing 15 μl PBS buffer solution. After being incubated for 20 min, 5 μl ‘jellyfish’ probe, 2 μl Exo III enzyme and 2 μl H1 probe were added to the mixture. The mixture was incubated at room temperature for 30 min. After adding 200 ml of 4 mM ABTS² and 1 ml of 30% H₂O₂, the reaction mixture was incubated at room temperature for 5 min. A UV-visible spectrophotometer was used to measure the mixture's absorbance at 418 nm and absorbance spectra between 500 and 400 nm compared with a blank.

Results & discussion

Working mechanism of Exo III-based colorimetric approach

Figure 1 illustrates the basic operation of the colorimetric assay for ultrasensitive CRP measurement by combining arch probe-based selective target recognition and ‘jellyfish’ probe-mediated triple-signal amplifications. The arch probe comprises two functional domains: one for CRP recognition built on the basis of an aptamer sequence and the other for signal amplification activation following CRP recognition. Due to the high affinity between CRP and the aptamer, the conformation of aptamer sequence changes in the presence of CRP and the aptamer disassociates from the arch probe, leaving the initiator sequence to activate subsequent signal amplification.

In the signal amplification procedure, a ‘jellyfish’ probe is created by hybridizing an ssDNA sequence on the loop part of a hairpin probe. The toehold region (a) hybridizes with the liberated initiator sequence making the ‘jellyfish’ probe have a 3′ blunt terminus. Exo III can therefore catalyze the sequential cleavage of mononucleotides in the 3′ direction, releasing the initiator to create a new cleavage process (cycle i). The resulting dsDNA product with two toeholds (b and e) unfolds the H1 probe and forms a 3′ blunt terminus (b terminus). Exo III continues to work down the ssDNA chain from the b section, cleaving the b and f sections, liberating the a′ section, H1 probe and other complementary chains containing domains c and e, among which a′, which shares the same sequence as the initiator sequence, and the H1 probe can serve as the new activator sequences to trigger continuous digestion (cycle ii, cycle iii). The ‘b+c’ sequence's G-quadruplex (b) allows for the formation of DNAzyme following the addition of hemin. The ABTS²-H₂O₂ system can be catalyzed by the DNAzyme acting as peroxidase mimics, amplifying the colorimetric observable signals.

Construction & feasibility of arch & ‘jellyfish’ probes

One requirement for the colorimetric technique was that the arch probe precisely recognize CRP and subsequently release the initiator sequence. The 3′ terminal of the aptamer sequence and the 5′ terminal of the initiator sequence were labeled with a FAM moiety and BHQ-1, respectively, to test the assembly of the arch probe and its feasibility for recognizing CRP. A high FAM signal was observed in the FAM-labeled aptamer sequence in Figure 2A. After assembly, the emission light of the FAM moiety was quenched by BHQ and the fluorescent signal significantly decreased. Upon the addition of CRP, the FAM signal recovered, indicating the initiator sequence disassociated from the arch probe. To evaluate the stability of the approach, the arch probe was used to detect CRP in different experimental environments, such as in PBS buffer, in BSA solution and in Dulbecco’s modified Eagle medium (DMEM), and the detection performances of the probe were evaluated. The recorded FAM signals of the arch probe when performed in PBS buffer, BSA solution and DMEM showed no statistical difference, indicating the high stability of the approach (Supplementary Figure 1). SYBR Green I was exploited to test the assembly of the ‘jellyfish’ probe. SYBR Green I is a dye that can emit green light when it binds to all double-helix groove regions in dsDNA. In Figure 2B, the recorded SYBR signal of the linear hairpin probe was lower compared with that of the assembled hairpin probe. A continuously enhanced signal was observed when the ‘jellyfish’ probe was constructed, implying that the ssDNA chain was hybridized on the loop section of the hairpin probe. The UV-visible absorbance spectra in the substrate solution containing hemin and ABTS^2--H₂O₂ were measured using 0.1 μg/ml CRP with or without Exo III and H1 probes to confirm the viability of the entire sensing procedure. The mixture devoid of CRP was studied as a control. Without the production of DNAzyme, hemin alone could only provide a poor catalytic efficiency toward ABTS^2--H₂O₂. This was shown by weak absorbance at 418 nm. Notably, when CRP, Exo III and H1 probes were present in the mixture of hemin and ABTS^2--H₂O₂, the absorbance significantly increased compared with the control, indicating that these three substances were necessary to create the DNAzyme and induce color reaction (Figure 2C).

Optimization of experimental parameters

The Exo III concentration and H1 probe were examined to improve the performance of the CRP detection method. Exo III enzyme at various doses (0–2.0 U/l) was incubated with 0.1 μg/ml CRP. According to Figure 3A, the Δ absorbance value (changes in absorbance with and without CRP) improved with increasing enzyme concentrations and reached a plateau at 1 U/l, indicating that 1 U/l Exo III provided a noticeably improved detection performance. The effect of the H1 probe on the detection performance of the approach was also investigated. The H1 probe concentration rose, causing the absorbance to rise and reach a relative plateau at 200 nM. (Figure 3B). As a result, 1 U/l Exo III and 200 nM H1 probe were selected to detect CRP at a higher absorbance value.

Analytical performance of colorimetric approach

The analytical performance of the colorimetric technique was assessed by detecting various concentrations of CRP in solutions containing 1% human serum (5, 50, 100, 500, 1000, 5000, 10,000 and 50,000 μg/ml) under the most favorable experimental circumstances. As the target CRP concentration increased, the absorbance rose (Figure 4A). A strong linear association was evident in the response versus logarithm of CRP concentration plot, with a correlation coefficient of 0.9971 (Figure 4B). Furthermore, the LOD using S/N = 3 was 1.32 ng/ml, which was significantly lower than the LOD using earlier published approaches (Supplementary Table 2). Moreover, the intraday and interday precisions of the CRP analyses were 1.78% (n = 10) and 3.33% (n = 10), respectively (Figure 4C). The method's specificity for CRP detection was also researched. Four distinct types of proteins were chosen as interfering substances and detected under the same circumstances (quantities of CRP, IL-6, AFP, TB and IgG were all 400 ng/ml). Figure 4D displays that only in the presence of CRP was the absorbance in the approach noticeably enhanced, indicating that the established colorimetric approach was more sensitive to CRP.

Conclusion

Elevated baseline CRP levels are associated with an increased risk of developing cardiovascular disease. CRP levels are significantly elevated in patients undergoing primary, elective CABG surgery. Considering the crucial role of CRP in the pathological process of acute-phase inflammatory and mediating inflammation reactions, we proposed a colorimetric approach for sensitive, reliable and direct CRP analysis, in hopes of facilitating the precise evaluation of CABG risk. The method contains two functional processes, including the CRP aptamer-based specific identification of CRP and ‘jellyfish’ probe-mediated Exo III-assisted signal amplification. The released initiator sequence in the CRP recognition process hybridized with the toehold section in the ‘jellyfish’ probe to activate Exo III-assisted triple-signal amplification processes, exposing the DNAzyme sequences (a well-known horseradish peroxidase mimic). The DNAzyme formation catalyzed the ABTS^2--H₂O₂-based color reaction. The approach exhibited a low LOD of 1.32 ng/ml, and high intraday and interday precisions for CRP detection. In conclusion, this method offers a sensitive and portable approach for CRP detection and, by modifying the aptamers, it shows promise for detecting more proteins. Considering the high sensitivity, simplicity and stability, the established approach could eventually be utilized in clinical molecular diagnostics and in evaluating inflammatory responses in patients receiving CPB.

Future perspective

CRP is a significant indicator to evaluate inflammatory responses. A sensitive and reliable analysis of CRP will greatly facilitate disease diagnosis. A colorimetric method via ‘jellyfish’ probe-based Exo III-assisted multiple-signal recycles for the detection of CRP was proposed. The method exhibited a low LOD of 1.32 ng/ml and high intraday and interday precisions for CRP detection. The merits of high sensitivity, simplicity and stability endow the established approach with promising prospects in the clinical diagnosis of diseases such as infection.