Exploiting Purine as an Internal Standard for SERS Quantification of Purine Derivative Molecules Released by Bacteria.

Surface-enhanced Raman scattering (SERS) is a highly sensitive technique used in diverse biomedical applications including rapid antibiotic susceptibility testing (AST). However, signal fluctuation in SERS, particularly the widespread of signals measured from different batches of SERS substrates, compromises its reliability and introduces potential errors in SERS-AST. In this study, we investigate the use of purine as an internal standard (IS) to recalibrate SERS signals and quantify the concentrations of two important purine derivatives, adenine and hypoxanthine, which are the most important biomarkers used in SERS-AST. Our findings demonstrate that purine IS effectively mitigates SERS signal fluctuations and enables accurate prediction of adenine and hypoxanthine concentrations across a wide range (5 orders of magnitude). Calibrations with purine as an IS outperform those without, exhibiting a 10-fold increase in predictive accuracy. Additionally, the calibration curve obtained from the first batch of SERS substrates remains effective for 64 additional substrates fabricated over a half-year period. Measurements of adenine and hypoxanthine concentrations in bacterial supernatants using SERS with purine IS closely align with the liquid chromatography-mass spectrometry results. The use of purine as an IS offers a simple and robust platform to enhance the speed and accuracy of SERS-AST, while also paving the way for in situ SERS quantification of purine derivatives released by bacteria under various stress conditions.

An antibiotic concentration gradient microfluidic device integrating surface-enhanced Raman spectroscopy for multiplex antimicrobial susceptibility testing.

Antimicrobial susceptibility testing (AST) is a key measure in clinical microbiology laboratories to enable appropriate antimicrobial administration. During an AST, the determination of the minimum inhibitory concentration (MIC) is an important step in which the bacterial responses to an antibiotic at a series of concentrations obtained in separate bacterial growth chambers or sites are compared. However, the preparation of different antibiotic concentrations is time-consuming and labor-intensive. In this paper, we present a microfluidic device that generates a concentration gradient for antibiotics that is produced by diffusion in the laminar flow regime along a series of lateral microwells to encapsulate bacteria for antibiotic treatment. All the AST preparation steps (including bacterium loading, antibiotic concentration generation, buffer washing, and isolated bacterial growth with an antibiotic) can be performed in a single chip. The viable bacterial cells in each microwell after the antibiotic treatment are then quantified by their surface-enhanced Raman scattering (SERS) signals that are acquired after placing a uniform SERS-active substrate in contact with all the microwells. For proof-of-concept, we demonstrated the AST performance of this system on ampicillin (AMP)-susceptible and -resistant E. coli strains. Compared with the parameters for conventional AST methods, the AST procedure based on this chip requires only 20 µL of bacteria solution and 5 h of operation time. This result indicates that this integrated system can greatly shorten and simplify the tedious and labor-intensive procedures required for current standard AST methods.

A microfluidic microwell device operated by the automated microfluidic control system for surface-enhanced Raman scattering-based antimicrobial susceptibility testing.

Bloodstream infection (BSI) is a serious public health issue worldwide. Timely and effective antibiotics for controlling infection are crucial towards patient outcomes. However, the current culture-based methods of identifying bacteria and antimicrobial susceptibility testing (AST) remain labor-intensive and time-consuming, and are unable to provide early support to physicians in critical hours. To improve the effectiveness of early antibiotic therapy, Surface-enhanced Raman scattering (SERS) technology, has been used in bacterial detection and AST based on its high specificity and label-free features. To simplify sample preparation steps in SERS-AST, we proposed an automated microfluidic control system to integrate all required procedures into a single device. Our preliminary results demonstrated the system can achieve on-chip reagent replacement, bacteria trapping, and buffer exchange. Finally, in-situ SERS-AST was performed within 3.5 h by loading isolates of ampicilin susceptible and resistant E. coli and clear discrimination of two strains under antibiotic treatment was demonstrated. Overall, our system can standardize and simplify the SERS-AST protocol and implicate parallel bacterial detection. This prototypical integration demonstrates timely microbiological support to optimize early antibiotic therapy for fighting bacteremia.

Bacteria encapsulation and rapid antibiotic susceptibility test using a microfluidic microwell device integrating surface-enhanced Raman scattering.

The antibiotic susceptibility test (AST) is a general laboratory procedure for bacterial identification and characterization and can be utilized to determine effective antimicrobials for individual patients. Due to the low bacterial concentration, conventional AST usually requires a prolonged bacterial culture time and a labor-intensive sample pretreatment process. Therefore, it cannot perform timely diagnosis or treatment, which results in a high mortality rate for seriously infected patients. To address this problem, we developed a microfluidic microwell device integrating surface-enhanced Raman scattering (SERS) technology, or the so called the Microwell-SERS system, to enable a rapid and high-throughput AST. Our results show that the Microwell-SERS system can successfully encapsulate bacteria in a miniaturized microwell with a greatly increased effective bacteria concentration, resulting in a shorter bacterial culture time. By attaching a microchannel onto the microwell, a smooth liquid and air exchange can purify the surrounding buffer and isolate bacteria in an individual microwell for independent SERS measurement. For proof-of-concept, we demonstrated a 2 h AST on susceptible and resistant E. coli and S. aureus with a concentration of 103 CFU mL-1 in the Microwell-SERS system, whereas the previous SERS-AST method required 108 CFU mL-1 bacterial suspension droplets dispensing on a SERS substrate. Based on the above features, we envision that the Microwell-SERS system could achieve highly sensitive, label-free, bacteria detection and rapid AST to enable timely and accurate bacterial infection disease diagnosis.

Antibiotic Susceptibility Test with Surface-Enhanced Raman Scattering in a Microfluidic System.

Antibiotic susceptibility test (AST) is essential in clinical diagnosis of serious bacterial infection, such as sepsis, while it typically takes 2-5 days for sample culture, antibiotic treatment, and reading result. Detecting metabolites secreted from bacteria with surface-enhanced Raman scattering (SERS) enables rapid determination of antibiotic susceptibility, reducing the AST time to 1-2 days. However, it still requires 1 day of culture time to obtain sufficient quantity of bacteria for sample washing, bacterial extraction, and antibiotic treatment. Additionally, the whole procedure, manually performed in open environment, often suffers from contamination and human error. To address the above problems, a microfluidic system integrating membrane filtration and the SERS-active substrate (MF-SERS) was developed to perform on-chip bacterial enrichment, metabolite collection, and in situ SERS measurements for antibiotic susceptibility test. Using Escherichia coli as the prototype bacterium, the lowest SERS detection limit of bacterial concentration of the MF-SERS system is 103 CFU/mL, which is 4 orders of magnitude lower than that using centrifugation-purification procedure, significantly shortening the bacterial culture time. The bacteria and secreted metabolites are enclosed during bacterial trapping, metabolite filtration, and SERS detection, thus minimizing possible contamination and human errors. Finally, the successful demonstration of AST on E. coli with a concentration of 103 CFU/mL is presented. Overall, the MF-SERS system with a miniature size and well-confined microenvironment allows the integration of multiple bacteria processes for bacterial enrichment, culture, and determination of AST.

Quantification of biomolecules responsible for biomarkers in the surface-enhanced Raman spectra of bacteria using liquid chromatography-mass spectrometry.

Recently, specific biomarkers in the surface-enhanced Raman scattering (SERS) spectra of bacteria have been successfully exploited for rapid bacterial antibiotic susceptibility testing (AST) - dubbed SERS-AST. The biomolecules responsible for these bacterial SERS biomarkers have been identified as several purine derivative metabolites involved in bacterial purine salvage pathways (W. R. Premasiri, J. C. Lee, A. Sauer-Budge, R. Theberge, C. E. Costello and L. D. Ziegler, Anal. Bioanal. Chem., 2016, 408, 4631). Here we quantified these metabolites in the SERS spectra of Staphylococcus aureus and Escherichia coli using ultra-performance liquid chromatography/electrospray ionization-mass spectrometry (UPLC/ESI-MS). The time dependences of the concentrations of these molecules were measured using 13C- or 12C-purine derivatives as internal and external standards respectively in UPLC/ESI-MS measurements. Surprisingly, a single S. aureus and an E. coli cell were found to release millions of adenine and hypoxanthine into a water environment in an hour respectively. Furthermore, simulated SERS spectra of bacterial supernatants based on the mixtures of purine derivatives with measured concentrations also show great similarity with those of the corresponding bacterial samples. Our results not only provide a quantitative foundation for the emerging SERS-AST method but also suggest the potential of exploiting SERS for in situ monitoring the changes in bacterial purine salvage processes in response to different physical and chemical challenges.

Au Nanoparticles Immobilized on Honeycomb-Like Polymeric Films for Surface-Enhanced Raman Scattering (SERS) Detection.

We have successfully developed novel surface-enhanced Raman scattering (SERS) substrates with three-dimensional (3D) porous structures for effectively improving the sensitivity and reproducibility of SERS, which can rapidly detect small molecules (rhodamine 6G as an example). Periodical arrays of the honeycomb-like substrates were fabricated by self-assembling polyurethane-co-azetidine-2,4-dione (PU-PAZ) polymers. PU-PAZ comprising amphiphilic dendrons could stabilize the phase separation between the water droplets and polymer solution, and then organize into regular porous structures during the breath figure method. Subsequently, SERS substrates were fabricated by immobilizing gold nanoparticles (AuNPs) onto the honeycomb-like films with various 3D porous structures, controlled by the different PU-PAZ concentrations and relative humidities. Results show that surface enhancement factors of honeycomb-like substrates were 20 times higher than that of flat-film substrates (control group) due to enormous hot-spots resonance effects by the 3D porous structure, verified through Raman mapping at various positions of the z-axis. Furthermore, the particle size effects were evaluated by immobilized 12 and 67 nm of AuNPs on the honeycomb-like substrates, indicating larger AuNPs could induce more pronounced hot-spots effects. The generation of hot-spots resonance to enhance Raman intensity is strongly dependent on the diameter of AuNPs and the pore size of the honeycomb-like and 3D porous substrates for label-free and rapid SERS detection.