Exploiting the Clinical Potential of a Chemiluminescent Probe for Mycoplasma pneumoniae Diagnosis

Abstract

Mycoplasma pneumoniae (MP) is a leading pathogen responsible for community-acquired pneumonia in the whole population, posing a life-threatening risk in severe disease. Current diagnostic methods, including culture, serology, and PCR, are limited by low sensitivity, delayed results, or operational complexity, highlighting the urgent need for rapid and reliable alternatives. Critically, MP metabolism produces hydrogen peroxide (H₂O₂), a key virulence factor, making it a promising biomarker for MP infection. Compared to other optical approaches, chemiluminescence-based one offers a powerful alternative for pathogen detection due to their high sensitivity, low cost, and rapid response. Herein, we report the first application of a chemiluminescent probe (MPCL) based on the established phenoxy-dioxetane scaffold with a 4-nitro-α-ketoamide trigger for direct MP detection. Our study demonstrated the practical value of this probe in distinguishing MP from other respiratory pathogens through quantitative H₂O₂ detection. MPCL enabled sensitive quantification and real-time imaging of H₂O₂ in MP-infected mice, and, when applied to clinical respiratory samples, achieved excellent diagnostic performance, with ROC analysis showing an area under the curve of 0.992 (95% CI: 0.974–1.000), 100% sensitivity, and 96.5% specificity. These results establish MPCL as a practical chemiluminescent tool with strong translational potential for rapid, low-cost, and reliable diagnosis of MP infections in both clinical and preclinical settings.

1. Introduction

Mycoplasma pneumoniae (MP) is a minimal prokaryotic microorganism characterized by the absence of a cell wall, high morphological variability, and the ability to pass through sterilizing filters. It is among the most common pathogens responsible for community-acquired pneumonia (CAP), accounting for 3% to 20% of cases, and up to 50% during epidemic peak years. , Clinical symptoms of MP infection are typically nonspecific, often presenting as mild and self-limiting. However, approximately 25% of cases can progress to severe pneumonia, potentially resulting in respiratory failure. , In the United States, an estimated 2 million MP infections occur annually, leading to approximately 100,000 hospitalizations among adults. − Given its high prevalence and clinical impact, timely and accurate diagnosis of MP is essential for early intervention and effective management of CAP.

Current diagnostic approaches for MP infection include pathogen culture, serological testing, and molecular methods. Although pathogen culture is considered the gold standard, it is rarely used in clinical practice due to its low sensitivity, specialized media requirements, technical complexity, and prolonged turnaround time of 2–4 weeks. , Serological testing remains the most widely used method in many primary care settings because of its convenience. However, its accuracy depends on a 4-fold increase in antibody titers between paired sera collected during the acute and convalescent phases, typically 2 weeks apart. As such, it is more suitable for epidemiological surveillance rather than timely outpatient diagnosis. − Molecular diagnostics, particularly real-time quantitative polymerase chain reaction (PCR), demonstrate high sensitivity and specificity and are widely applied in clinical laboratories. Nonetheless, these techniques involve complex protocols, costly equipment, and the need for trained personnel, limiting their use in resource-limited or point-of-care settings. , Given these limitations, there is an urgent need for rapid, reliable, user-friendly, and cost-effective diagnostic tools for MP detection.

As a wall-less bacterium, MP relies primarily on phospholipids and their metabolites, such as glycerol, as its main sources of carbon and energy. A key enzyme in this process is l-α-glycerophosphate oxidase (GlpO), which is exposed on the pathogen surface and plays a central role in glycerol metabolism. GlpO catalyzes the oxidation of l-α-glycerophosphate (Glp) into dihydroxyacetone phosphate and H₂O₂, contributing to both energy production and the pathogen’s virulence through H₂O₂ generation. − The resulting H₂O₂, along with reactive oxygen species (ROS) generated by host cells, contributes to oxidative stress in respiratory epithelial tissues. Therefore, H₂O₂, as a metabolic product of MP, represents a promising diagnostic marker for MP pneumonia.

Some fluorescent molecules and fluorescence-based imaging strategies have been developed for the detection of H₂O₂. However, these approaches rely on external light excitation, which can cause autofluorescence and photon scattering from animal tissues, thereby compromising detection accuracy. − In contrast, chemiluminescence (CL) probes function without external excitation, thereby eliminating autofluorescence interference and enabling high sensitivity with low background noise. , These advantages post chemiluminescence as a promising approach for biosensing and bioimaging. Despite these benefits, many conventional chemiluminescent systems have notable limitations. For instance, acridinium ester and luminol-based probes require alkaline conditions, while luciferin/luciferase systems depend on genetically modified organisms expressing luciferase. − Recently, chemiluminescent platforms based on dioxetane scaffolds have emerged as powerful tools for both in vitro and in vivo imaging due to their enhanced sensitivity, favorable tissue penetration, and low biological toxicity. , It has been reported that the 4-nitro-α-ketoamide group undergoes nucleophilic attack by H₂O₂, followed by Baeyer–Villiger rearrangement and hydrolysis to yield p-nitrobenzoic acid, carbon dioxide, and an amine. , Based on this mechanism, we incorporated the 4-nitro-α-ketoamide moiety into a phenoxy-dioxetane scaffold to synthesize a chemiluminescent probe responsive to H₂O₂. This represents the first application of this scaffold/trigger combination for direct clinical diagnosis of MP infection. MPCL enables both quantitative detection and real-time imaging of H₂O₂ in complex biological environments and holds strong potential for the early diagnosis of MP infection.

2. Experimental Sections

2.1. Synthesis and Characterization of MPCL

MPCL was synthesized according to the route outlined in Scheme by using compound 1 and compound 2 as precursors.

1. Synthesis and Proposed Mechanism of MPCL Responses to H₂O₂ .

^a Experimental conditions: (i) TEA, DMF, DCM; (ii) methylene blue, O₂, hυ, DCM.

Under a nitrogen atmosphere, compound 1 (50 mg, 1.0 equiv) was dissolved in 1 mL of dichloromethane (DCM), followed by the addition of catalytic amounts of N, N-dimethylformamide (DMF) and triethylamine (TEA, 40 mg, 3.0 equiv). The mixture was stirred at room temperature for 1 h. Compound 2 (47 mg, 1.0 equiv) was then added, and the reaction was allowed to proceed with continuous stirring. Progress was monitored by thin-layer chromatography (TLC), and the reaction was completed after 6 h. After that, 20 mL of ethyl acetate was added to the reaction mixture, and the pH was adjusted to neutral using a saturated ammonium chloride (NH₄Cl) solution. The mixture was washed three times with saturated brine, and the organic phase was collected. The solvent was removed under reduced pressure to afford a yellow solid. The crude product was dissolved in 1 mL of DCM, and methylene blue (5 mg) was added as a photosensitizer. The solution was stirred at room temperature under an oxygen atmosphere and yellow light irradiation for 8 h. TLC confirmed completion of the reaction. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1, v/v) to yield MPCL as a yellow solid (42.3 mg, 46.7% yield). ¹H NMR (600 MHz, CDCl₃) δ 9.0 (s, 1H), 8.6 (d, J = 8.5 Hz, 2H), 8.4 (d, J = 8.6 Hz, 2H), 8.0–7.9 (m, 2H), 7.7 (d, J = 8.2 Hz, 2H), 7.6 (d, J = 8.3 Hz, 1H), 7.5 (d, J = 8.2 Hz, 2H), 6.5 (d, J = 16.2 Hz, 1H), 4.9 (s, 2H), 3.8 (s, 3H), 3.2 (s, 3H), 2.3 (d, J = 13.1 Hz, 1H), 2.0 (d, J = 3.6 Hz, 1H), 1.9 (dd, J = 25.3, 13.1 Hz, 2H), 1.8–1.7 (m, 3H), 1.7–1.6 (m, 5H), 1.5 (dt, J = 13.1, 2.9 Hz, 1H), 1.3 (tt, J = 16.4, 7.8 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 185.9, 171.2, 166.8, 157.7, 154.1, 151.0, 138.2, 137.6, 136.6, 135.3, 133.1, 132.6, 131.6, 130.0, 129.3, 129.1, 127.7, 125.3, 123.6, 121.2, 120.1, 111.7, 96.4, 75.6, 60.4, 51.9, 49.7, 36.6, 33.9, 33.6, 32.7, 32.2, 31.6, 31.5, 26.2, 25.8, 21.1, 14.2. MS (ES⁺): m/z calcd for C₃₇H₃₅ClN₂O₁₀: 702.1980; found: 725.1877 [M + Na]⁺ (Figures S1–S3).

2.2. Cell Culture and Cytotoxicity Studies

A549 cells [ATCC CCL-185] were cultured in T25 culture dishes containing 10% fetal bovine serum albumin and 1% Dulbecco’s modified Eagle medium (DMEM), then incubated at 37 °C at a CO₂ concentration of 5%. The medium containing the A549 cells was then pretransferred to a 96-well plate and cultured at 37 °C for 12 h. To study its cytotoxicity, different concentrations of MPCL (i.e., 0, 20, 40, 60, 80, and 100 μM) were then added to each well and incubation was continued for a further 12 h. Subsequently, an aliquot (20 μL) of the CCK-8 solution was added to each well and incubated for 2 h. Following that, the absorbance of each well was recorded at 490 nm using a microplate reader.

2.3. Pathogen Culture

The standard MP strain ATCC 29342 (MP-S) and a clinical isolate obtained from Minhang Hospital (MP-C), Fudan University were cultured in SP4 medium at 37 °C in a 5% CO₂ atmosphere for 7 days prior to use in subsequent experiments.

Moraxella catarrhalis (MC), Staphylococcus aureus (S. aureus), Serratia marcescens (SM), Klebsiella pneumoniae (KP), and Escherichia coli (E. coli)also collected from the same clinical sourcewere cultured in brain heart infusion (BHI) broth under identical conditions (37 °C, 5% CO₂, 12 h) for comparative studies.

2.4. Establishment and Validation of MP Infection Mouse Model

All animal studies were conducted in accordance with the protocol approved by the Experimental Animal Ethics Committee of School of Pharmacy, Fudan University. BALB/C mice (Female, 5–6 weeks old, Shanghai Lingchang Biotechnology Co., Ltd.) were used for the animal experiments. After acquisition, the mice were acclimated in an animal room maintained at 22–25 °C with 50–60% relative humidity for 1 week. Following isoflurane anesthesia, the mice were intranasally administrated with MP suspension (100 μL, 1 × 10⁷ CFU/mL) daily for five consecutive days. The MP infection model was established and then used for the subsequent experiments.

The MP infection mouse model was characterized by H&E staining of lung tissues and measurement of macrophages in mouse alveolar lavage fluid through flow cytometry analysis on markers of CD11b and F4/80.

2.5. Verification of the Clinical Application of MP Chemiluminescent Probes

Between July 2024 and September 2025, a total of 51 clinical nasopharyngeal swab samples were collected. Among them, 29 were PCR-negative (PCR^–), including samples without detectable pathogen infection (n = 24), single infections with KP (n = 3), S. aureus (n = 1), and E. coli (n = 1). The remaining 22 samples were PCR-positive (PCR⁺) (KP, S. aureus and E. coli were identified by culture, while MP were identified by Nucleic Acid Detection Kit for Six Respiratory Pathogens, Sansure Biotech Inc., China) for the validation of clinical utility of MPCL. This study was approved by Ethics Committee of Minhang Hospital, Fudan University (No. 2024-036-01K), and informed consent was obtained from all participants.

3. Results and Discussion

3.1. Design on Chemiluminescent Probe for H₂O₂ and MP Sensing

Compared with fluorescent probes, 1,2-dioxetane-based chemiluminescent probes do not require external light excitation. Instead, they emit light through energy released from internal chemical reactions, making them suitable for detecting H₂O₂ under complex physiological and pathological conditions. The design of the reactive group plays a key role in determining probe performance. Introducing a strong electron-withdrawing phenylnitro group to the α-ketoamide structure enhances reactivity toward H₂O₂ and significantly boosts luminescent emission, thereby improving sensitivity. Based on this strategy, we developed a chemiluminescent probe incorporating a 4-nitro-α-ketoamide moiety as the H₂O₂ recognition unit, enabling real-time tracking of H₂O₂ fluctuations during MP metabolism. In the probe’s stable form, the phenolic hydroxyl group is masked by the 4-nitro-α-ketoamide moiety, preventing background luminescence. The reaction mechanism is shown in the Scheme , upon exposure to H₂O₂, the α-ketoamide undergoes nucleophilic attack by hydroperoxide anions, followed by a Baeyer–Villiger rearrangement and subsequent hydrolysis to yield carboxylic acid (Figure S4), carbon dioxide, and an amine. Concurrently, the self-immolative group (p-aminobenzyl alcohol) is cleaved, releasing the phenolic hydroxyl anion (Figure S5). This triggers an intramolecular charge transfer process which produces a strong chemiluminescence signal.

3.2. Optical and Chemical Properties of MPCL

After synthesizing the probe, we investigated the chemiluminescence kinetics of MPCL (20 mM, pH = 7.4, 1% DMSO) in response to increasing concentrations of H₂O₂. As shown in Figure A, the chemiluminescence intensity progressively increased over time, reaching a maximum value at approximately 80 min. Notably, the intensity of the chemiluminescent signal correlated positively with H₂O₂ concentration. Within the range of 5–30 μM H₂O₂, the signal exhibited a strong linear relationship with concentration (Y = 2.176X – 0.2008, R ² = 0.9970, Figure B), demonstrating the probe’s excellent potential for real-time imaging of H₂O₂ under physiological conditions.

1.

Optical and chemical properties of probe MPCL. (A) Kinetic profiles of MPCL (20 μM) incubated with H₂O₂ in a range of concentrations (5–30 μM); (B) linear correlation between CL intensity of MPCL and H₂O₂ concentration; (C) CL response of MPCL incubated with different substances including metal cations (1. blank, 2. K⁺, 3. Ca²⁺, 4. Mg²⁺, 5. Al³⁺, 6. Cu²⁺, 7. Cys, 8. Tyr, 9. Lys, 10. Val, 11. Ala, 12. Arg, 13. ROO·, 14. ¹O₂, 15. ClO^–, 16. OH, 17. ONOO^–, 18. H₂O₂). Data were expressed as mean ± SD (n = 3); (D) CL intensity of MPCL (20 μM) after incubation with H₂O₂ (100 μM) at different pH; (E) fluorescence emission spectrum of MPCL (20 μM) before (black) and after (green) incubation with H₂O₂ (10 μg/mL); (F) cell viability of A549 cells treated with MPCL (0–100 μM).

To further investigate the specificity of MPCL to H₂O₂, we then examined the effects of other potential coexisting substances including metal cations (1 mM, K⁺, Ca²⁺, Mg²⁺, Al³⁺, Cu²⁺), amino acids (1 mM, Cys, Tyr, Lys, Val, Ala, Arg) and ROS (200 μM, ROO·, ¹O₂, ClO^–, ·OH, ONOO^–, H₂O₂) on the probe. As shown in Figure C, only H₂O₂ can initiate the chemiluminescence of MPCL without interference from other ROS. To evaluate the effect of pH on the reactivity of the H₂O₂ probe, we then monitored the reaction of MPCL with H₂O₂ in solutions of different pH (Figure D). The probe (20 μM, 2% DMSO, saline) was stored in the dark under a pH range (3, 4, 5, 6, 7, 7.4, 8, 9, and 10, adjusted with 1 M HCl or NaOH) at 37 °C for 1 h. Afterward, they were incubated with H₂O₂ (100 μM) and the chemiluminescence intensity was detected. Strong chemical signals were observed in acidic or alkaline pH media (pH 5.0–9.0). Also, the probe also exhibited good stability under different temperature conditions (Figure S6).

After completing the chemiluminescence experiments, we measured the fluorescence emission spectrum of MPCL degradation byproducts (20 μM, pH 7.4, 1% DMSO). As shown in Figure E, MPCL exhibited a weak emission peak at 550 nm in the absence of H₂O₂. Upon the addition of H₂O₂, a marked change in the fluorescence emission spectrum was observed, with a significant enhancement of fluorescence intensity at 550 nm. This fluorescence enhancement was consistent with previously reported behavior of similar dioxetane-based chemiluminescent systems, further confirming the probe’s responsiveness to H₂O₂. In addition, after incubation of 100 μM MPCL with A549 cells for 2 h, the cell viability remained above 80%, demonstrating the good cell compatibility of the probe (Figure F). These data suggested that MPCL has the potential to sensitively detect H₂O₂ in vivo.

3.3. Assessment of Pathogen Metabolic Hydrogen Peroxide Levels

As described, MPCL demonstrates excellent stability, high specificity, superior sensitivity and low cytotoxicity, enabling the reliable detection of H₂O₂ levels in biological systems. To assess its potential for diagnosing MP infection, we first collected several common pathogens, including MC, S. aureus, SM, KP, and E. coli.

Following pathogen proliferation, cultures were centrifuged to remove the supernatant and washed twice with HEPES buffer (containing 67.6 mM HEPES, 140 mM NaCl and 7 mM MgCl₂, pH 7.4). The pathogen pellets were resuspended in 1.5 mL HEPES buffer, transferred to new tubes, and centrifuged to remove the supernatant (10 min, 10,000 rpm, 4 °C). The resulting precipitates were washed and finally resuspended in 4 mL HEPES buffer. Cell density was adjusted to an absorbance of 1.0 at 550 nm (A_{5 5 0}). The samples were incubated at 37 °C for 1 h, followed by the addition of glycerol (final concentration: 100 μM) and a further 2-h incubation at 37 °C.

We then evaluated the ability of MPCL to monitor pathogen H₂O₂ production in vitro. As shown in Figure , H₂O₂ levels produced by both clinical and standard MP strains (MP-C and MP-S) were comparable without obvious difference, but significantly higher than those produced by other pathogen species. The results suggest that MPCL can effectively distinguish MP from other common pathogens based on H₂O₂ levels, offering a promising strategy for preliminary screening and early diagnosis.

2.

Assessment of pathogen H₂O₂ production levels, including MC, S. aureus, SM, KP, E. coli, MP-C and MP-S. Student’s t tests were used to evaluate statistically significant differences between groups. ***P < 0.001, ****P < 0.0001. ns, not significant.

3.4. Evaluation of MP Infection Mouse Model

Encouraged by the findings above, we further investigated the potential of MPCL for the in vivo detection of MP respiratory infections. MP infection models were established in C57BL/6 mice by intranasal administration of MP suspension once daily for five consecutive days. To confirm successful model establishment, the mice were sacrificed on day six, and both lung tissues and bronchoalveolar lavage fluid (BALF) were collected for analysis. As shown in Figure A, macrophage counts in BALF were significantly elevated, confirming the successful induction of MP infection. Additionally, histopathological examination (H&E) revealed pulmonary interstitial edema and substantial inflammatory cell infiltration in the alveolar spaces (Figure B).

3.

Evaluation of MPCL for monitoring H₂O₂ level in MP infection mouse model. (A) Measurement of neutrophils in mouse alveolar lavage fluid through flow cytometry based on the markers of CD11b and F4/80; (B) H&E staining image of lung tissues; (C) luminescence images of MP and vehicle groups mice after inhalation of MPCL; (D) quantification of part (C); (E) luminescence intensity of MP and vehicle groups at 35 min. Data are expressed as mean ± SD (n = 4). Student’s t tests were used to evaluate statistically significant differences between groups. *P < 0.05.

Following confirmation, mice were anesthetized and administered MPCL intranasally (20 μL, 80 μM). Chemiluminescence imaging of the nasopharyngeal region was then captured. As anticipated, mice in the MP-infected group exhibited a rapid and intense chemiluminescent response, in contrast to the vehicle group (Figure C). This signal was time-dependent, peaking at approximately 185 min. For practical purposes, the signal intensity at 35 min was selected for quantitative comparison (Figure D). The elevated signal in infected mice is attributed to increased H₂O₂ production by metabolically active MP.

These results collectively demonstrated that MPCL enabled sensitive and noninvasive detection of MP infection through in vivo chemiluminescence, highlighting its promise as an alternative diagnostic tool.

3.5. Clinical Validation of Probe MPCL for MP Evaluation

A total of 51 clinical nasopharyngeal swab samples were collected between July 2024 and September 2025, including 22 PCR+ and 29 PCR- cases. Among the PCR- specimens, 24 showed no detectable pathogen infection, while the remaining included single infections with KP (n = 3), S. aureus (n = 1), and E. coli (n = 1). These samples were incubated with the MPCL probe, and the resulting chemiluminescence intensity was measured. The results demonstrated a strong concordance between the MPCL probe and PCR-based detection of MP, with no significant statistical difference between the two methods (Figure A). In further, a statistically significant difference was observed between PCR-positive and PCR-negative groups by using the MPCL method (P < 0.0001). Receiver operating characteristic (ROC) curve analysis yielded an area under the curve (AUC) of 0.992 (95% CI: 0.974–1.000), indicating excellent diagnostic performance, with a sensitivity of 100% and a specificity of 96.5% (Figure B). These findings highlight the high accuracy of the MPCL probe in clinical sample detection and support its promise as a reliable, efficient auxiliary tool for diagnosing MP infections. Unlike PCR, the MPCL method does not require complex instrumentation or specialized personnel, further underscoring its practical value in clinical settings.

4.

Evaluation of clinical nasopharyngeal specimens. (A) CL intensity of MPCL in 51 clinical respiratory samples, including 22 PCR+ and 29 PCR–; (B) ROC curve analysis based (A). Student’s t tests were used to evaluate statistically significant differences between groups. ****P < 0.0001.

4. Conclusions

In this study, we integrated the 4-nitro-α-ketoamide trigger into a phenoxy-dioxetane scaffold to construct a chemiluminescent probe (MPCL) for selective H₂O₂ detection. MPCL enables sensitive and real-time quantification of H₂O₂ produced by MP metabolism, providing a robust tool for dynamic monitoring of pathogen activity. Importantly, the probe demonstrates high selectivity for MP over other pathogen species and shows strong diagnostic performance in patient-derived clinical samples. These results highlight the translational potential of MPCL as a rapid, noninvasive, and practical diagnostic tool for early detection of MP infections, supporting timely clinical decision-making and potential evaluation of therapeutic interventions.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/cbmi.5c00096.

• NMR, MS spectra, and thermal stability of MPCL (PDF)

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Y.J., Z.C., and Y.L. are contributed equally to this paper.

The authors affirm that this manuscript is an original work and has not been submitted or published elsewhere. The authors have adhered to the ethical guidelines outlined in the publication process, and any use of previously published material has been appropriately cited.

The authors declare no competing financial interest.