Flexible PDMS-SERS platform for culture-free diagnosis of bacterial infections in clinical wound care

Author links open overlay panelYanhong Zhou a b 1,Wei Zhang a b 1,Shaowen Cheng a b 1,Yanlong Xing a b,Juan Wang c,Fabiao Yu a b,Rui Wang a b

• aKey Laboratory of Emergency and Trauma, Ministry of Education, Key Laboratory of Hainan Trauma and Disaster Rescue, Key Laboratory of Haikou Trauma, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, 571199, China

• bEngineering Research Center for Hainan Bio-Smart Materials and Bio-Medical Devices, Key Laboratory of Hainan Functional Materials and Molecular Imaging, College of Emergency and Trauma, Hainan Medical University, Haikou, 571199, China

• cKey Laboratory of Tropical Translational Medicine, Ministry of Education, Key Laboratory of Brain Science Research and Transformation in Tropical Environment of Hainan Province, School of Basic Medicine and Life Sciences, Hainan Medical University, Haikou, 571199, China

Received 20 December 2024, Revised 31 March 2025, Accepted 2 April 2025, Available online 3 April 2025, Version of Record 8 April 2025.

https://doi.org/10.1016/j.talanta.2025.128089

Highlights

• •SERS platform was developed for culture-free detection of three bacteria.

  •The platform demonstrated low LODs and monitored bacterial count changes.

• •The platform successfully detected bacteria in clinical samples.

• Abstract

The early and accurate detection of pathogenic bacteria in infected wounds is crucial for preventing severe complications such as sepsis and multiple organ failure. This study presents a new surface-enhanced Raman scattering (SERS) platform based on polydimethylsiloxane (PDMS) flexible substrates and SERS probes for culture-free detection of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa). The PDMS flexible substrates, functionalized with gold nanoparticles (Au NPs) and specific antibodies, facilitate the efficient capture of bacteria and enhance the SERS signals through electromagnetic field coupling. The SERS probes, composed of Au@Ag core-shell nanoparticles with double-layer Raman reporter molecules, provide high sensitivity and specificity in bacterial detection. In the presence of target bacteria, the immunocomplex of PDMS flexible substrates, bacteria, and SERS probes forms, generating strong SERS signals and enabling sensitive bacterial detection. The SERS platform demonstrated limits of detection as low as 2.79 CFU/mL for E. coli, 3.42 CFU/mL for S. aureus, and 4.52 CFU/mL for P. aeruginosa. Additionally, the platform successfully monitored bacterial count changes in infected wound models and detected bacteria in clinical samples. These results indicate that the proposed SERS platform is a powerful tool for the rapid and sensitive detection of multiple bacteria in infected wounds, offering significant potential for clinical applications in infection management and advancing the field of bacterial diagnostics.

Graphical abstract

SERS imaging method for the sensitive and selective detection of bacteria based on SERS probes and PDMS flexible substrate.

Keywords

Surface-enhanced Raman scattering (SERS) imagingFlexible substratePathogenic bacteria detectionInfected woundClinical application

1. Introduction

Pathogenic microorganisms invade the body through wounds, causing damage to normal functions, metabolism, and tissue structure, leading to wound infections. Wound infection can cause various complications, such as sepsis, shock, and multiple organ failure, which not only increase the economic burden of patients but also threaten their lives [[1], [2], [3], [4]]. Therefore, early diagnosis and treatment of wound infections to prevent the occurrence of complications is of great significance for improving the prognosis of patients. The most common pathogens in bacterial infections are Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa). E. coli is facultative anaerobic bacterium, mostly non-pathogenic, and crucial for maintaining normal intestinal functions [5]. However, through the action of pathogenic islands and mobile genetic elements, some E. coli strains can obtain new virulence factors to trigger infections and diseases [6,7]. S. aureus widely exists in human skin, nasal cavity, oral cavity, intestinal tract and other parts [8,9]. It can use a variety of pathogenic factors to cause infection when the host is injured, burned, or immunosuppressed [10,11]. S. aureus can also produce β-lactamases, making it resistant to various antibiotics, which poses a challenge for the treatment of infections caused by S. aureus [[12], [13], [14], [15]]. P. aeruginosa is a widely distributed opportunistic pathogen that can cause various clinical infections, severe infectious diseases, and long-term chronic diseases even at very low concentration, especially affecting immunocompromised patients or patients admitted to intensive care units [16,17]. Therefore, early diagnosis can reduce the risk of systemic infection, avoid life-threatening complications such as sepsis, and provide important clinical guideline, helping doctors develop more accurate and effective treatment plans.Currently, bacteria colony counting is the gold standard for bacteria detection, which may require a pre-enrichment step up to 48 h [18,19]. Real-time polymerase chain reaction (RT-PCR) has been widely utilized for the microbiological identification, however, the sample pretreatment also needs long time [20,21]. Furthermore, the amplification of unrelated gene sequences often induces false positive interference. The critical number of bacterial infections is 103–105 CFU per gram of tissue or milliliter of body fluid, especially in the early stages of wound infection where the number of bacteria is relatively small, making it difficult to accurately detect under culture free conditions using bacteria colony counting and RT-PCR. Therefore, there is still an urgent need to develop new technique for highly sensitive and reliable detection of the bacteria on the infected wound under culture-free conditions.Surface-enhanced Raman scattering (SERS) has been attracted increasing attentions in detecting trace targets due to its ultrahigh sensitivity, non-invasive measurement and multiple detection capability [[22], [23], [24]]. The excellent performance of SERS in trace detection mainly depends on the active substrates, which can produce “hot spots” due to the strong localized surface plasmon resonance and greatly enhance the Raman signals of the targets or Raman reporter molecules [[25], [26], [27]]. Thus, a variety of SERS active substrates have been fabricated to improve the detection sensitivity. Among them, 3D SERS substrates could efficiently increase the number of hot spots and provide the uniform and consistent signals. Unfortunately, most of 3D substrates use the rigid materials, which can't be suitable for the in site bacteria detection on the infected wound due to the complex surface. On the contrast, the flexible substrate materials have gained increasing attentions because of the close contact with the complex surface. Flexible polymer membrane such as PDMS has been widely used for the development of smart flexible devices, which exhibits high tensile strength and strong adhesion [28,29]. In addition, PDMS molecules have very small Raman signals, meaning the scarce interference for the target detection. Importantly, the homogeneity of Au/Ag nanoparticles (Au/Ag NPs) immobilized on the PDMS membrane can significantly affect the SERS signals [30]. Therefore, the self-assembly transfer method has been utilized to fabricate the PDMS flexible substrates with homogeneous and dense gold nanoparticles. Furthermore, PDMS flexible substrates could efficiently capture the target bacteria after conjugation of specific antibodies on the surface of Au NPs layer.In this work, we developed a new SERS platform based on PDMS flexible substrates and SERS probes for highly sensitive detection of E. coli, S. aureus and P. aeruginosa on the infected wounds. PDMS flexible substrates played two vital roles in SERS signal enhancement due to the junctions between Au NPs layers and SERS probes and efficient capture of triple bacteria based on the specific antibodies. SERS probes were made of Au@Ag core-shell nanoparticles with Raman reporter double-layer on the surface of Au core and Ag shell, which further conjugated the detection antibodies through the functional linker (HS-PEG-COOH). In the presence of the target bacteria, the sandwich immunocomplexes could be formed among PDMS flexible substrates, bacteria and SERS probes. In addition, the quantitative analysis of E. coli, S. aureus and P. aeruginosa was performed through SERS imaging method based on the Raman signal intensities from the reporter molecules. With these features, the SERS platform was applied to monitor the count number changes of bacteria on the infected wounds and clinical samples. We believe that the proposed SERS platform based on PDMS flexible substrates and SERS probes opens a new opportunity for highly sensitive detection of bacteria on the infected wounds.

Fig. 4

The preparation protocol and characterization of PDMS flexible substrate. (A) The preparation procedure of PDMS flexible substrate based on paste-peel off method. (B) SEM of PDMS flexible substrate. (C) SERS spectra of 81 random points on the surface of PDMS flexible substrate. (D) SERS imaging of PDMS film after addition of SERS probes. (E) SERS imaging of the PDMS flexible substrate with immobilization of SERS probes. The above SERS imaging was achieved using exposure time of 1.0 s (633 nm-laser, 50× objective). (F–H) Statistical analysis of (D) and (E) was performed using one-way analysis of variance followed by post hoc Tukey multiple comparisons (n = 3, mean ± S. E. M., ∗P < 0.05, ∗∗P < 0.01).

Fig. 5. The specificity of SERS platform based on PDMS flexible substrate for culture-free detection of E. coli, S. aureus and P. aeruginosa. (A) The detection protocol of SERS platform. (B) SERS mapping images of Substrate-EC and E. coli after incubation with three SERS probes, respectively. (D) SERS mapping images of Substrate-SA and S. aureus after incubation with three SERS probes, respectively. (F) SERS mapping images of Substrate-PA and P. aeruginosa after incubation with three SERS probes, respectively. (C), (E) and (G) Statistical analysis of (B), (D) and (F) was performed using one-way analysis of variance followed by post hoc Tukey multiple comparisons (n = 3, mean ± S. E. M., ∗∗∗∗P < 0.0001), respectively.

Fig. 6. (A) SERS mapping images of E. coli, S. aureus and P. aeruginosa at various concentrations using SERS platform based on PDMS flexible substrate. (B), (C) and (D) Corresponding calibration line of Raman intensity at 1616 cm−1, 1646 cm−1 and 1133 cm−1 as a function of the logistic of the concentration of E. coli, S. aureus and P. aeruginosa, respectively. (E) Four-parameter logistic function for the fitting curve.

Fig. 7. (A) Schematic illustration of SERS platform for evaluation the count changes of E. coli, S. aureus and P. aeruginosa on the infected wounds. The illustration was created with the help of BioRender.com. (B) SERS imaging indicating the count changes of E. coli, S. aureus and P. aeruginosa, respectively. The photographs of PDMS flexible substrates coating on the infected wound. (C), (D) and (E) Quantification of normalized Raman signal intensity of E. coli, S. aureus and P. aeruginosa, respectively. (F) Colony formation plates in the wound model. (G)–(I) Plate colony forming unit counts of the mice model with E. coli, S. aureus and P. aeruginosa, respectively.

.9. Clinical sample tests

To further investigate the SERS platform's potential for culture-free bacteria detection, clinical samples from infected tissues were tested. Herein, the skin tissues at the lesion sites from four patients were collected for testing. The obtained fresh tissues were coated with PDMS flexible substrates for 2 h. SERS probes were dropped and incubated for SERS imaging. As shown in Fig. 8A, only the samples of patient 1 and patient exhibited strong SERS signals to indicate E. coli and S aureus infection on the wound, respectively, while the samples of patient 3 and patient 4 didn't show any SERS signals, demonstrating the absence of triple bacteria. On the other hand, the clinical test confirmed the E. coli and S. aureus infection of patient 1 and patient 2, respectively, as shown in Fig. 8B. Although patient 3 and patient 4 suffered from S. haemolyticus and Aspergillus, respectively, the extremely low SERS signal indicated the high specificity of the proposed SERS platform. All the results revealed that the SERS platform could be used for triple bacteria detection in the infected wounds.

1. Fig. 8 (A) SERS imaging of the bacteria on the skin infected tissues from four patients. (B) Comparation between SERS imaging method and clinical test.

4. Conclusion

In summary, we developed a new SERS-imaging platform for culture-free detection of E. coli, S. aureus and P. aeruginosa utilizing SERS probes and PDMS flexible substrates. On the one hand, the SERS probes, composed of Au@Ag core-shell nanoparticles with double-layered Raman reporter molecules, were conjugated with specific antibodies to enhance detection sensitivity. On the other hand, The PDMS flexible substrates, functionalized with gold nanoparticles and specific antibodies, efficiently captured bacteria and significantly enhanced SERS signals through electromagnetic coupling. This platform achieved detection limits of 2.79 CFU/mL for E. coli, 3.42 CFU/mL for S. aureus, and 4.52 CFU/mL for P. aeruginosa, demonstrating a sensitivity approximately two to three orders of magnitude higher than existing ELISA methods. In addition, the proposed SERS platform was successfully utilized to monitor bacterial count changes on infected wounds in animal models and to detect bacteria in clinical samples. These results highlighted the platform's potential for real-time, highly sensitive detection of bacterial infections, offering a significant advancement in the field of bacterial diagnostics. We believe that this SERS platform could broaden the application of SERS imaging in biomedical research and clinical practice.

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