博文
SERS nanoplatform for clinical detection of S. aureus
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aKey Laboratory of Hainan Trauma and Disaster Rescue, 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 Emergency and Trauma, Ministry of Education, Key Laboratory of Hainan Functional Materials and Molecular Imaging, College of Emergency and Trauma, Hainan Medical University, Haikou, 571199, China
cSchool of Pharmacy, Hainan Medical University, Haikou, 571199, China
dJiangyin Center for Disease Control and Prevention, No. 158 Changjiang Road, Jiangyin, 214431, China
Received 11 April 2024, Revised 30 July 2024, Accepted 7 August 2024, Available online 8 August 2024, Version of Record 15 August 2024.
https://doi.org/10.1016/j.talanta.2024.126691
Highlights•The aptamer-conjugated Fe3O4 magnetic nanoparticles and vancomycin modified-Au nanoparticles were successfully developed.
•SERS nanoplatform based on dual-recognition of aptamer and vancomycin was developed for the highly sensitive detection of S. aureus.
•The limit of detection of the proposed assay was found to be 3.27 CFU/mL with the range of 0–107 CFU/mL.
Abstract
Staphylococcus aureus (S. aureus) is the most common pathogen in human purulent infections, which can cause local purulent infections, as well as pneumonia, pseudomembranous enteritis, pericarditis, and even systemic infections. The conventional methods including bacteria colony counting, polymerase chain reaction and enzyme-linked immunosorbent assay can't fully meet the requirement of highly sensitive detection of S. aureus due to their own disadvantages. Therefore, it's an urgent need to develop new platform to detect S. aureus in the early infection stage. In this study, a new surface-enhanced Raman scattering (SERS)-based nanoplatform based on dual-recognition of aptamer (Apt) and vancomycin (Van) was developed for the highly sensitive detection of S. aureus. The SERS nanoplatform consisted of two functional parts: aptamer-conjugated Fe3O4 magnetic nanoparticles (Fe3O4-Apt MNPs) for bacteria enrichment and vancomycin modified-Au nanoparticles (Van-Au NPs) as the SERS probes for S. aureus quantitative detection. Upon the target bacteria enrichment, the SERS signals of the supernatant after magnetic separation could be obtained and analyzed under different concentrations of S. aureus. The limit of detection of the proposed assay was found to be 3.27 CFU/mL. We believe that the proposed SERS-based nanoplatform has great potential as a powerful tool in the early detection of specific bacteria.
Graphical abstract
Keywords Surface-enhanced Raman scattering (SERS) S. aureus detection Dual-recognition Aptamer and vancomycin
1. Introduction
Staphylococcus aureus (S. aureus) is one kind of the most common opportunistic pathogens affecting human survival and is considered to be one of the five leading pathogens associated with infection-related health [1,2]. It can infect almost all tissues, resulting in a variety of diseases including bacteremia, skin and soft tissue infection, ostemyelitis, pneumonia and so on [3,4]. Conventionally, bacterial culture and biochemical identification are considered as the gold standard in the detection of S. aureus [5]. However, it needs long culture time even several days due to the pre-enrichment and selective differential plating steps, which limits the rapid detection for clinical diagnosis [6,7]. Polymerase chain reaction (PCR) has been widely used as a powerful tool for bacteria identification; unfortunately, it still needs sample pretreatment procedures including DNA extraction and signal amplification by a sequential thermo-cycling process, which easily induce false-positive signals [8]. Although the enzyme-linked immunosorbent assay (ELISA) holds a variety of advantages including easy use, high stability and repeatability, the repeated incubation and washing steps are time-consuming and lacks in sensitivity and specificity [9,10]. Therefore, there is still an urgent need for rapid and specific detection of bacterial pathogens.
Recently, surface-enhanced Raman scattering (SERS) has attracted increasing attention due to its high sensitivity, simple operation and fingerprint spectra, which has been extensively used for the detection of proteins, DNA, bacteria, small molecules and toxic metal ions [11,12]. Two major types of SERS detection, label-free and label-based strategies, have been extensively applied for bacteria identification [13,14]. The former strategy is to directly obtain the Raman spectra of whole-organism fingerprint of bacteria through the close attachment of bacteria cell to SERS active substrates [15]. However, this method is seriously affected by complex matrix and lacks enough sensitivity [16]. In contrast, labeled-based detection is an indirect approach that using SERS probes as quantitative reporters for bacterial detection [17]. More interestingly, the combination of separation tools with SERS probes including magnetic approach, microfluidics and lateral flow assay, can efficiently improve the sensitivity of SERS detection and enrichment of target bacteria in complex samples [18,19]. In addition, one of the key challenges in these methods is to improve the binding efficiency of SERS probes toward the bacteria. Therefore, SERS probes with high sensitivity and specificity are crucial for SERS-based bacterial detection.
Nowadays, SERS probes-bacteria-substrates sandwich immunocomplex is the most common strategy in SERS-based immunoassay with a pair of antibodies or aptamers for recognizing the different antigenic epitopes on the bacteria surface [20,21]. However, the unspecific absorption induced by the cross-linking between antibodies/aptamers and other interferent usually induces the false signals. Thus, the dual-recognition strategy based on different moieties maybe the potential solution to overcome the above obstacle [[22], [23], [24], [25]]. In addition, the volume of immunocomplex is relatively large, making it difficult for the laser spot to fully cover, resulting in significant errors in the detection results. If the volume ratio among SERS probes, bacteria and substrates can be precisely regulated, the quantitative detection of the supernatant separated from the immunocomplex has the great potential to significantly reduce the fluctuations of SERS signals, which will efficiently improve the sensitivity and stability.
Here, we reported a universal SERS-based system with dual-recognition moieties of aptamers and vancomycin (Van) for highly sensitive and specific detection of S. aureus by integrating magnetic capture/separation and SERS probes (Van modification) binding. The proposed assay consisted of two steps performed simply in microtubes. The target bacteria were firstly captured and separated from the samples through aptamers conjugated Fe3O4 MNPs. Then, SERS probes were introduced to quantify the captured bacteria, which could specifically bind the gram-positive bacteria due to the presence of Van. Finally, the SERS signals for supernatant were recorded through Raman system. With the increasing concentration of S. aureus, more bacteria could be captured by Fe3O4 MNPs and recognized by SERS probes, resulting in the decreased SERS signals induced by the less SERS probes in the supernatant. The detection limit of S. aureus could reach 3.27 CFU/mL, which was much lower than that of the conventional methods. Moreover, the SERS detection method demonstrated high sensitivity for real clinical samples. We believe the proposed method could serve as a powerful detection system for diagnosis of bacterial infection.
Fig. 1. Schematic illustration of detect S. aureus via SERS-based nanoplatform. (A) Preparation of SERS detection probes. (B) Synthesis of magnetic separation platform. (C) The clinical application of SERS-based nanoplatform.
Fig. 2. Preparation and general characterization of Fe3O4-Apt MNPs and SERS probes. (A) Schematic diagram of the synthesis of Fe3O4-Apt MNPs. (B) Preparation procedure of SERS probes. (C) DLS size of Au NPs, Au-M NPs, and Van-Au NPs. (D) UV–vis absorption spectra of Au NPs, Au-M-PEG-NHS NPs, and Van-Au NPs. (E) Raman spectra of Au-M NPs and Van-Au NPs. (F) The cumulative Raman spectra changes within 14 days. (G) Stability test of SERS probes, the Raman signals was examined on day 1, day 7 and day 14 (n = 100). (H) and (I) TEM image of Au NPs and Van-Au NPs, respectively, scale bar: 50 nm.
Fig. 3. Recognition of SERS probes to S. aureus. TEM images of SERS probes and Au NPs after interaction with S. aureus, respectively, scale bar: 1 μm. Representative SERS mapping images at 1613 cm−1 of the SERS probe and Au NPs with S. aureus, scale bar: 5 μm.
Fig. 4. Capture performance of Fe3O4-Apt MNPs for S. aureus. (A) Representative bacterial colony formation photographs of Fe3O4-Apt MNPs capturing S. aureus at 15 min, 30 min, 45 min, and 60 min. Images of bacterial colony formation captured by Fe3O4 MNPs at 45 min are shown on the right. (B) Capture rates of S. aureus corresponding to (A). (C) Random microscopic images of S. aureus captured by Fe3O4-Apt MNPs, scale bar: 5 μm. (D) OD600 results for total bacterial suspension and supernatant. Experiments were repeated three times and presented as mean ± standard deviation (SD).
Fig. 5. Detection of S. aureus by SERS nanoplatform. (A) The supernatant photograph after incubation. The bacterial concentrations from left to right were 107, 106, 105, 104, 103, 102, 101, 0 CFU/mL (B) UV–visible absorption spectra of the supernatant after magnetic separation in A. (C) Raman spectra of the supernatant after magnetic separation in A, with characteristic peak position of 1613 cm−1 marked in blue. (D) Fitting curve of Raman signal intensity at 1613 cm−1 after treatment with different concentrations of S. aureus.
Table 1. Recovery experiment of S. aureus in PBS (n = 3).
| Sample NO | Concentration of S. aureu (CFU/mL) | Recovery rate (%) | Standard deviation (%) |
|---|---|---|---|
| 1 | 2 × 106 | 92.1 % | 8.1 % |
| 2 | 2 × 105 | 94.6 % | 4.2 % |
| 3 | 2 × 104 | 92.8 % | 6.9 % |
Fig. 6
Table 2. Recovery experiment of S. aureus in urine sample (n = 3).
| Sample NO | Concentration of S. aureu (CFU/mL) | Recovery rate (%) | Standard deviation (%) |
|---|---|---|---|
| 1 | 2 × 106 | 99.4 % | 10.3 % |
| 2 | 2 × 105 | 97.6 % | 4.9 % |
| 3 | 2 × 104 | 92.4 % | 6.9 % |
4. Conclusion
In summary, we developed a highly sensitive and dual-recognition SERS platform for detection of S. aureus. The formed SERS probes exhibited uniform size and stable SERS signal. The Fe3O4-Apt MNPs using as the substrate could achieve excellent capture efficiency on S. aureus. The SERS signal of the supernatant showed a negative relationship between the bacterial concentration and SERS signal intensity with the limit of detection of 3.27 CFU/mL. Urine bacterial samples could also be detected and quantified in this platform with satisfactory recovery. This SERS nanoplatform based on aptamers has the advantages of simple processing, low dosage and high detection sensitivity, which is expected to be a promising method for rapid and sensitive detection of bacteria.
https://blog.sciencenet.cn/blog-2438823-1446777.html
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