ENGINEERING

Cardiovascular diseases (CVDs) remain the leading cause of death worldwide. Nitric oxide (NO), a key biomarker closely associated with vascular function and the progression of CVDs, exhibits dynamic changes that hold significant pathophysiological implications [1]. As an endogenous gaseous signaling molecule, NO is involved in various physiological processes such as regulating vasodilation, maintaining blood pressure homeostasis, and modulating inflammatory responses [2-4].

Clinical studies have demonstrated that abnormal expression levels of NO are significantly correlated with the development and progression of major diseases including hypertension, atherosclerosis, and heart failure. However, direct and dynamic monitoring of NO in blood remains challenging due to its short half-life, low concentration in vivo, and high reactivity with substances such as oxygen radicals.

Current commonly used NO detection methods—such as the Griess assay, electrochemical sensing, and electron spin resonance (ESR)—are often labor-intensive, rely on bulky instruments, and are unsuitable for real-time in situ detection, limiting their application in rapid clinical diagnostics and point-of-care testing (POCT). Therefore, there is an urgent need in the biomedical detection field to develop a highly sensitive, specific, and user-friendly NO detection tool for quantitative analysis in complex biological fluids such as blood.

Design

In this study, the NO-binding domain of NorR [5] was selected as the recognition module and fused to yellow fluorescent protein (YFP), red fluorescent protein (mCherry), and green fluorescent protein (GFP) via a flexible linker. Upon NO binding to the NorR domain, the induced conformational change alters the optical properties of the fluorescent protein through an allosteric effect, thereby converting NO concentration into a quantifiable fluorescent signal.

This strategy provides an effective means for highly specific and real-time detection of NO in complex biological environments. We propose to produce the fluorescent probes using a prokaryotic expression system in E. coli. The anticipated experimental workflow is shown in Fig.1.

Fig.1 Gene circuit for prokaryotic expression

Build

We constructed the prokaryotic expression vectors using a double restriction enzyme digestion strategy, as outlined in Fig. 2. First, the DNA fragments encoding YFP, mCherry, and GFP were amplified by PCR. Both the PCR products and the pRSETa-NorR vector were digested with the restriction enzymes BamH I and KpnI. The digested DNA fragments were purified and ligated into the linearized vector using T4 DNA ligase, resulting in the recombinant plasmids NorR-YFP_pRSETa, NorR-mCherry_pRSETa, and NorR-GFP_pRSETa. These constructs were then transformed into E. coli DH5α competent cells. Positive clones were identified by colony PCR (Fig. 4), and selected clones were cultured for plasmid extraction and subsequent sequencing.

Fig.2 Workflow diagram of plasmid construction

Fig.3 Vector construction of NorR-YFP_pRSETa, NorR-mCherry_pRSETa and NorR-GFP_pRSETa

A: PCR of YFP sequences; B: PCR of mCherry sequence; C: PCR of GFP sequences; D: The pRSETa vector after double enzyme digestion.

Fig.4 PCR Verification of the recombinant plasmid

Test

1. Protein expression and purification

Based on the successfully constructed recombinant plasmids, the NorR-fluorescent protein fusion was expressed in E. coli BL21(DE3) cells. Soluble expression was achieved through low-temperature induction with 0.42 mM IPTG. After cell lysis, the fusion protein was purified by nickel affinity chromatography via its His-tag, and high-purity protein fractions were obtained using gradient imidazole elution (Fig. 5). These purified samples were subsequently used for further studies.

Fig.5 The purification results of recombinant proteins NorR-YFP, NorR-GFP and NorR-mCherry

2. The influence of different fluorescent protein carriers on the sensitivity of nitric oxide (NO) probes

To evaluate the NO-binding capabilities of YFP, GFP, and mCherry, each probe was incubated with varying concentrations of the NO donor Furoxan, and their specific fluorescence signals were detected. YFP and GFP were measured in dual-excitation mode (excitation at 420 nm and 485 nm, emission at 515 nm) to determine the fluorescence ratio, while mCherry was detected under 590 nm excitation and 645 nm emission. The binding performance was quantitatively assessed by calculating the ratio of fluorescence intensity after NO binding to that before binding. As shown in Fig. 6, with increasing Furoxan concentration, the fluorescence ratios of YFP and GFP changed significantly and gradually saturated, demonstrating a concentration-dependent response. In contrast, the fluorescence ratio of mCherry showed no notable change across the concentration range, indicating its inability to effectively bind NO. Further comparison revealed that YFP exhibited a significantly higher response amplitude than GFP, indicating superior sensitivity. Therefore, YFP was selected as the NO-sensing probe for subsequent experiments.

Fig.6 Fluorescence response curves of the YFP-, GFP- and mCherry-NO probes

3. Verification of the NO sensing function of NorR-YFP

3.1 pH value and temperature stability

We further evaluated the stability of the protein under different temperatures and pH conditions. Each 100 μL protein sample was treated with 100 μM Furoxan (NO donor) in the experimental group, while the control group received an equal volume of buffer. As shown in Fig. 7, after NO release induced by Furoxan, the fluorescence ratio of NorR-YFP at 420/480 nm increased significantly. The protein exhibited a stable fluorescence response within the pH range of 7.2–8.0 and the temperature range of 20–40°C, demonstrating excellent environmental adaptability and detection reliability, making it suitable for NO detection in complex physiological systems.

Fig.7 pH value and temperature stability of NorR-YFP

3.2 Specific detection of NO

To assess the specificity of NorR-YFP, we examined its fluorescence response in the presence of various reactive molecules. The system was treated with 100 μM of Furoxan (NO donor), CO, H₂O₂, NaNO₂, or NaHS, and changes in the fluorescence ratio were monitored. The results (Fig. 8) showed that only Furoxan treatment induced a rapid and significant increase in the fluorescence ratio, while other reactive molecules caused no notable signal changes. This confirms that NorR-YFP possesses highly selective recognition capability for NO and can effectively distinguish NO from other similar reactive small molecules.

Fig.8 Specificity assessment of NorR-YFP toward various reactive species

3.3 Concentration-dependent detection

To elucidate the dose-response relationship between the fluorescence signal of NorR-YFP and NO concentration, the protein was treated with gradient concentrations of the NO donor Furoxan. The experimental system contained 100 μL of 1 μmol/L protein sample, and changes in the fluorescence ratio were continuously monitored over 40 minutes. The results showed that as the Furoxan concentration increased, the fluorescence response of NorR-YFP enhanced in a clear concentration-dependent manner (Fig. 9), fully demonstrating that the fusion protein can produce a sensitive dose-dependent response to NO.

Fig.9 Real-time Monitoring of NorR-YFP Fluorescence Kinetics upon Furoxan Stimulation

3.4 Dependence of Nitric Oxide Concentration on Fluorescence Detection in Artificial Plasma

In the plasma NO detection experiment, different concentrations (0–1000 μM) of the NO donor Furoxan were incubated with artificial plasma and the probe protein. After blue light excitation, the fluorescence ratio significantly increased with rising Furoxan concentration, showing a clear dose-dependent relationship (Fig. 10). The results indicate that the probe can sensitively and reliably detect NO in plasma, laying the foundation for quantitative analysis of NO in biological samples.

Fig.10 The dose-dependent response of probe protein fluorescence intensity in artificial plasma to the concentration of NO donor furoxan

3.5 Subcellular localization detection

To achieve specific monitoring of NO at the subcellular level, lentiviral vectors targeting different cellular regions were synthesized: mito-NorR-YFP_pLVX (mitochondria), cyto-NorR-YFP_pLVX (cytoplasm), and nuclear-NorR-YFP_pLVX (nucleus). At 48 hours after transfection of these vectors into 293T cells, fluorescence microscopy observations (Fig. 11) revealed effective expression of the NorR-YFP fusion protein in the cells, with fluorescence distributions fully consistent with the expected localizations: a reticular or punctate pattern in the mitochondria group, uniform diffusion in the cytoplasm group, and concentration in the nuclear region in the nucleus group. These results indicate that the constructed probes possess accurate subcellular targeting ability, providing a reliable tool for real-time and specific monitoring of NO dynamics in specific organelles.

Fig.11 The subcellular localization of the NO sensor NorR-YFP

Learn

This study successfully developed a novel NO biosensor based on the NorR recognition module. The NorR-YFP fusion protein demonstrated high sensitivity, excellent environmental stability, and specificity, enabling real-time quantitative detection of NO under physiologically relevant conditions.

It performed well in artificial plasma and various subcellular structures. This sensor provides a powerful tool for mechanistic research and clinical diagnosis of cardiovascular diseases.

In the future, this study is expected to further advance the development of in vivo real-time imaging technology and portable NO detection devices, offering innovative solutions for precision medicine and point-of-care diagnostics.

Design

Based on the successful construction of the original NorR-YFP fusion protein in the first cycle, we conducted a detailed analysis of its potential limitations. We hypothesized that the length of the Linker connecting the NorR sensing domain and the YFP reporter domain is a key but yet unoptimized parameter that affects the performance of the sensor. An inappropriate Linker length may lead to spatial steric hindrance between the domains or a low efficiency in conformational change transmission.

Therefore, we have designed a series of Linker variants, aiming to systematically study the influence of Linker length on the performance of the sensor. The specific strategy is to construct a set of variants that are shorter and longer than the original Linker, in order to find the optimal length that can maximize the signal response amplitude. The detailed DNA sequences of all the designed Linkers are shown in the Tab.1.

Build

To construct the aforementioned Linker variants, we employed the double restriction enzyme digestion method. We used specific restriction endonucleases to digest the vector backbone and the insertion fragments containing different Linker sequences, then purified, ligated, and transformed the ligated products into competent cells. However, after screening colonies by colony PCR and DNA sequencing verification on a large number of transformants, the results indicated that no correct recombinant plasmids of any variant could be obtained. All sequencing results showed phenomena such as vector self-ligation, incorrect insertion, or non-specific recombination, indicating a systematic failure in the cloning process.

Test - Not Completed

Due to the failure to obtain any correct plasmid constructs during the "construction" phase, the subsequent protein expression, purification, and functional testing of this project cannot be carried out.

Learn

Although this cycle failed to obtain functional Linker variants due to the failure in constructing double-cutting vectors, the work at this stage verified at the design level the significant potential of optimizing the Linker system to enhance the performance of the NorR-YFP probe. The gradient variants designed based on the Linker length provided a clear experimental foundation for the precise regulation of the sensor's performance. This technical bottleneck also prompted us to recognize that adopting more efficient cloning methods (such as Golden Gate assembly) is crucial for advancing such optimization studies. In the future, after overcoming the construction obstacles, the targeted modification of the Linker region will remain a core strategy for enhancing the probe's sensitivity and stability, and it is worthy of being pursued as a key direction.