Solid-phase extraction coupled to automated centrifugal microfluidics SERS: Improving quantification of therapeutic drugs in human serum.

Surface-enhanced Raman spectroscopy (SERS) is a powerful method in analytical chemistry, but its application in real-life medical settings has been limited due to technical challenges. In this work, we introduce an innovative approach that is meant to advance the automation of microfluidics SERS to improve reproducibility and label-free quantification of two widely used therapeutic drugs, methotrexate (MTX) and lamotrigine (LTG), in human serum. Our methodology involves a miniaturized solid-phase extraction (µ-SPE) method coupled to a centrifugal microfluidics disc with incorporated SERS substrates (CD-SERS). The CD-SERS platform enables simultaneous controlled sample wetting and accurate SERS mapping. Together with the assay we implemented a machine learning method based on Partial Least Squares Regression (PLSR) for robust data analysis and drug quantification. The results indicate that combining µ-SPE with CD-SERS (µ-SPE to CD-SERS) led to a substantial improvement in the signal-to-noise ratio compared to combining CD-SERS with ultrafiltration or protein precipitation. The PLSR model enabled us to obtain the limit of detection and quantification for MTX as 2.90 and 8.92 µM, respectively, and for LTG as 10.76 and 32.29 µM. We also validated our µ-SPE to CD-SERS method for MTX against HPLC and immunoassay (p-value <0.05), using patient samples undergoing MTX therapy. In addition, we achieved a satisfactory recovery rate (80%) for LTG when quantifying it in patient samples. Our results show the potential of this newly developed approach as a strategy for therapeutic drugs in point-of-care clinical settings and highlight the benefits of automating label-free SERS assays.

APELLA: Open-Source, miniaturized All-in-One powered Lab-on-a-Disc platform.

We present an unconventional approach to a common Lab-on-a-Disc (LoD) that combines a quadcopter propulsion system, a miniaturized 2.4 GHz Wi-Fi spy camera, 9.74 Watt Qi wireless power, and an Arduino into an open-source, miniaturized All-in-one powered lab-on-disc platform (APELLA). The quadcopter propulsion generates thrust to rotate (from 0.1 to 24.5 Hz) or shake the LoD device, while the spy camera enables a real-time (30 frames per second) and high definition (1280 × 720 pixels) visualization of microfluidic channels without requiring a bulky and heavy stroboscopic imaging setup. A mobile device can communicate with an Arduino microcontroller inside the APELLA through a Bluetooth interface for closed loop and sequential frequency control. In a proof-of-concept study, the APELLA achieved comparable mixing efficiency to a traditional spin stand and can capture rapid microfluidic events at low rotational frequencies (<5Hz). The APELLA is low-cost (c.a. 100 Euro), compact (15.6 × 15.6 × 10 cm3), lightweight (0.59 kg), portable (powered by a 5 V USB power bank), and energy efficient (uses < 6% power of the conventional system), making it ideal for field deployment, education, resource-limited labs.

Moving perfusion culture and live-cell imaging from lab to disc: proof of concept toxicity assay with AI-based image analysis.

In vitro, cell-based assays are essential in diagnostics and drug development. There are ongoing efforts to establish new technologies that enable real-time detection of cell-drug interaction during culture under flow conditions. Our compact (10 × 10 × 8.5 cm) cell culture and microscope on disc (CMoD) platform aims to decrease the application barriers of existing lab-on-a-chip (LoC) approaches. For the first time in a centrifugal device, (i) cells were cultured for up to six days while a spindle motor facilitated culture medium perfusion, and (ii) an onboard microscope enabled live bright-field imaging of cells while the data wirelessly transmitted to a computer. The quantification of cells from the acquired images was done using artificial intelligence (AI) software. After optimization, the obtained cell viability data from the AI-based image analysis proved to correlate well with data collected from commonly used image analysis software. The CMoD was also suitable for conducting a proof-of-concept toxicity assay with HeLa cells under continuous flow. The half-maximal inhibitory time (IT50) for various concentrations of doxorubicin (DOX) in the case of HeLa cells in flow, was shown to be lower than the IT50 obtained from a static cytotoxicity assay, indicating a faster onset of cell death in flow. The CMoD proved to be easy to handle, enabled cell culture and monitoring without assistance, and is a promising tool for examining the dynamic processes of cells in real-time assays.

Enhanced Eradication of Mucin-Embedded Bacterial Biofilm by Locally Delivered Antibiotics in Functionalized Microcontainers.

Bacterial biofilm-related infections are difficult to eradicate and require repeated treatments with high doses of antibiotics. Thus, there is an urgent need for new treatment strategies that minimize the use of antibiotics while enhancing biofilm eradication. Functionalized reservoir-based microdevices, such as, microcontainers (MCs), offer, high drug loading capacity, mucus embedment, and tuneable drug release. Here, MCs are loaded with the antibiotic ciprofloxacin (CIP), and sealed with a lid consisting of chitosan (CHI) and a mucolytic agent, N-acetylcysteine (NAC). It is found that CHI and NAC work synergistically, showing improved mucoadhesive and mucolytic properties. To better mimic the in vivo habitat of Pseudomonas aeruginosa (P. aeruginosa), the biofilm is grown in a mucin-containing medium on a newly developed centrifugal microfluidic system. The CHI/NAC coated MCs improve eradication of biofilm (88.22 ± 2.89%) compared to CHI-coated MCs (72.68 ± 3.73%) or bolus injection (39.86 ± 13.28%). The findings suggest that MCs are significantly more efficient than a bolus treatment. Furthermore, CHI/NAC functionalized MCs kill most of the biomass already after 5 h (80.75 ± 3.50%), mainly due to a fast drug release. This is the first time that CHI/NAC has been combined as a coating to explore mucolytic properties on bacterial biofilms.

Bacterial Cell Cultures in a Lab-on-a-Disc: A Simple and Versatile Tool for Quantification of Antibiotic Treatment Efficacy.

Pathogenic bacterial biofilms can be life-threatening, greatly decrease patient's quality of life, and are a substantial burden on the healthcare system. Current methods for evaluation of antibacterial treatments in clinics and in vitro systems used in drug development and screening either do not facilitate biofilm formation or are cumbersome to operate, need large reagent volumes, and are costly, limiting their usability. To address these issues, this work presents the development of a robust in vitro cell culture platform compatible with confocal microscopy. The platform shaped as a compact disc facilitates long-term bacterial culture without external pumps and tubing and can be operated for several days without additional liquid handling. As an example, Pseudomonas aeruginosa biofilm is grown from single cells, and it is shown that (1) the platform delivers reproducible and reliable results; (2) growth is dependent on flow rate and growth medium composition; and (3) efficacy of antibiotic treatment depends on the formed biofilm. This platform enables biofilm growth, quantification, and treatment as in a conventional flow setup while decreasing the application barrier of lab-on-chip systems. It provides an easy-to-use, affordable option for end users working with cell culturing in relation to, e.g., diagnostics and drug screening.

Injection molded lab-on-a-disc platform for screening of genetically modified E. coli using liquid-liquid extraction and surface enhanced Raman scattering.

We present the development of an automated centrifugal microfluidic platform with integrated sample pre-treatment (filtration and liquid-liquid extraction) and detection (SERS-based sensing). The platform consists of eight calibration and four assay modules, fabricated with polypropylene using injection molding and bonded with ultrasonic welding. The platform was used for detection of a secondary bacterial metabolite (p-coumaric acid) from bacterial supernatant. The obtained extraction efficiency was comparable to values obtained in batch experiments and the SERS-based sensing showed a good correlation with HPLC analysis.