In Situ Detection of Endotoxin in Bacteriostatic Process by SERS Chip Integrated Array Microchambers within Bioscaffold Nanostructures and SERS Tags

Abstract

In order to achieve real-time and in situ detection of endotoxin, which is an important and significant clinical test index, surface-enhanced Raman spectroscopy (SERS) chip integrated array microchambers within bioscaffold nanostructures and a SERS monitoring strategy were proposed in this paper. After sputtering of nanogold on the cicada wing, which was selected as a natural template, and polydimethylsiloxane bonding, array-type chambers within bioscaffold nanostructures were prepared for in situ bacterial culture and monitoring of endotoxin in the bacteriostasis process by SERS. Meanwhile, the SERS tag modified with the DNA aptamer was prepared and added into this complex biochemical reaction to further improve the sensitivity and selectivity. A new method for in situ detection of endotoxin was thus established. The detection time was shortened to 100 s, and the detection limit was as low as 6.25 ng/mL. Pseudomonas aeruginosa was cultured in situ in the chamber of the SERS chip with antimicrobial agents in 0−72 h. The endotoxin released in the antibacterial process was monitored by the designed SERS detection strategy. The results obtained by SERS analysis were consistent with those of the ELISA kit.

SERS Sensing of Bacterial Endotoxin on Gold Nanoparticles

“…However, the SERS system detects the signal from the few NPs available in the laser area -that is about 1000 AuNPsproviding a real LOD of 160 (± 10) fg/mL, that is lower than the detection limit of LAL assays (13). Furthermore, the proposed SERS approach does not suffer the limit of the fluorophore quenching, which is a serious issue in colorimetric and fluorogenic LPS detection assays, especially for nanoparticles, and allows to measure the number of LPS molecules directly on the single AuNP, without the use of SERS tag or functionalization procedures, as previously reported (21). Indeed, since the number of LPS molecules adsorbed on the NPs, in saturation condition, can be evaluated by the DLS measurements, as reported in the Supplementary Table 1, it is possible to estimate the SERS SNR as a function of the number of LPS molecules per AuNP (47).…”

“…Therefore, the AuNPs allows direct sensing of LPS, providing a good enhancement and reproducibility of the signal and avoiding the use of Raman reporters as previously reported (21). At the same time, the proposed approach allows for noninvasive and non-destructive characterization of the local chemical structure of the molecules attached to the AuNPs, avoiding the use of functionalization procedures.…”

“…Therefore, due to the Raman fingerprint, SERS-based sensing allows to identify and quantify molecules with excellent sensitivity and reproducibility in different environments, thereby enabling the use of the SERS technique for numerous biomedical and biosensing applications (16)(17)(18)(19)(20). Indirect SERS sensing, based on the combined use of a SERS tag and a selective aptamer-based binding, has been demonstrated for quantitative detection of endotoxins in a solution (21). Additional advantages of the SERS technique are that it does not require labelling or other specific treatments for sample preparation, measurements can be performed on few mL of desired NP concentration, and the technique is non-destructive, allowing for performing multiple measurements on the same sample.…”

Environmental protection based on the nanobiosensing of bacterial lipopolysaccharides (LPSs): material and method overview

“…The outcomes received with the aid of using SERS evaluation have been steady with the ones of the ELISA kit. 130 An innovative work describes the use of SERS for the label-free quantitative nanoscale identification of the LPS adsorbed on the surface of 50 nm AuNPs. This SERS method was an efficient tool to detect LPS on the AuNP surface and the basis for the development of a novel specific and sensitive LPS-detection sensor based on the use of SERS and AuNPs.…”

Highly Sensitive Detection and Differentiation of Endotoxins Derived from Bacterial Pathogens by Surface-Enhanced Raman Scattering

“…Recent ultra-sensitive SERS-based methods can detect single molecule reactions on a substrate surface [12,13]; however, Raman-based studies on the unique spectra of bacterial endotoxin are currently very limited. A recent study reported the in situ detection of a Pseudomonas aeruginosa endotoxin using a nanogoldsputtered cicada wing as a SERS chip; however, it used 4-mercaptobenzoic acid (4-MBA) and p-aminophenol (PAP) as SERS reporters rather than the intrinsic SERS spectra of the endotoxin itself [14]. In order to establish a highly sensitive SERS platform for intrinsic endotoxin detection, highly reproducible and practical substrates are essential.…”