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Clinical on-site monitoring of ß-lactam antibiotics for a personalized antibiotherapy

An appropriate antibiotherapy is crucial for the safety and recovery of patients. Depending on the clinical conditions of patients, the required dose to effectively eradicate an infection may vary. An inadequate dosing not only reduces the efficacy of the antibiotic, but also promotes the emergence of antimicrobial resistances. Therefore, a personalized therapy is of great interest for improved patients’ outcome and will reduce in long-term the prevalence of multidrug-resistances. In this context, on-site monitoring of the antibiotic blood concentration is fundamental to facilitate an individual adjustment of the antibiotherapy. Herein, we present a bioinspired approach for the bedside monitoring of free accessible ß-lactam antibiotics, including penicillins (piperacillin) and cephalosporins (cefuroxime and cefazolin) in untreated plasma samples. The introduced system combines a disposable microfluidic chip with a naturally occurring penicillin-binding protein, resulting in a high-performance platform, capable of gauging very low antibiotic concentrations (less than 6 ng ml−1) from only 1 µl of serum. The system’s applicability to a personalized antibiotherapy was successfully demonstrated by monitoring the pharmacokinetics of patients, treated with ß-lactam antibiotics, undergoing surgery.


Proof of principle testing of the designed bioassay on a microtiter plate
Stop-flow measurement technique S2: Proof of principle and feasibility of the designed bioassay: Calibration curve of ampicillin performed on a microtiter plate (Costar cat. no. 3590) and optically read-out. 10 µg ml −1 of PBP3 in 50 mM bicarbonate buffer pH 9.6 was coated overnight at 4 °C on the surface of the microtiter plate. After a 1 h blocking step (300 µl of 1 % BSA in PBS buffer), the sample mixed with conjugate was added and incubated for 1 h, followed by 100 µl of 1 µg ml −1 avidin-GOx for 30 min. Finally 100 µl of substrate solution (PBS buffer containing, 40 mM glucose, 375 µg ml −1 ABTS and 40 ng ml −1 HRP) was added to the well. The enzymatic reaction was followed via the measurement of product formation by spectrophotometry at 405 nm for 30 minutes.

S3:
Illustration of the applied stop-flow protocol for 6 different analyte concentrations. a) A flow of 40 mM glucose solution at a rate of 20 µl min −1 was applied. During the stop-phase (1, 2 or 5 min), the enzyme's production of H2O2 goes further on. By restarting the flow, the accumulated H2O2 is flushed through the electrochemical cell, where the hydrogen peroxide is electrochemically detected, resulting in an on-chip calibration curve b).

Optimization of the blocking method and time
Performance characteristics of the microfluidic platform S4: Comparison of different blocking strategies after 15 min immobilization of 1 µg ml −1 GOx labeled avidin, followed by a 1 min stop-flow readout technique with 40 mM glucose solution. All blocking methods, except the overnight blocking, were applied for 1 hour. The best blocking efficiency for glucose oxidase labeled avidin was achieved with overnight blocking of 1 % BSA solution. However, an incubation time of 1 hour was used for the assay protocol, since there was no significant improvement of the blocking efficacy compared to the overnight blocking procedure. The error bars show the standard deviation of two parallel measurements. To estimate the costs per single biosensor, the calculations are based on the fabrication of 460 wafers. For the total cost of a single sensor, each material and fabrication step is considered, including the costs for the polyimide substrate, the platinum pattern, the DFR layers and the chemical reagents like SU-8, silver and silver chloride electrolytes as well as developer and remover solutions. The estimated material costs per single chip are listed in table S8.
S8: Estimated costs of the fabrication materials per single biosensor.

Materials
Costs Due to the low reagent consumption of the biosensor of less than 1 µl, the needed volumes for the immunoassay are also very low. For the coating of the surface with antibodies, a concentration of 200 µg ml −1 is used. Therefore, roughly 1250 biosensors can be coated with a stock antibody solution of 125 µl (at 2 mg ml −1 ). Since the antibody solution is priced at about 600 € per mg, the antibody costs per single chip 0.12 €. Using similar calculations for the other used biomolecules, wash buffer and 1 % BSA blocking solution, the total costs for all reagents per single chip are about 0.13 €. Thus, the overall costs of the microfluidic device for the personalized antibiotherapy is calculated to be only 0.62 € per sample.
For the fabrication process of the DFR based biosensor, a total work time of 10 hours (excluding the Pt pattern and the hard bake times in the oven) is estimated. By terms of automation, the work time can be reduced, regarding the degree of automation of the process. Furthermore, the assay preparation, for example the antibody immobilization, can be even further more automated, since now every reagent is dispensed by hand. However, it is possible to combine the here employed system with a robotic technique for spotting multiple biomolecules on individual chip inlets in parallel, which increases the throughput and reduces therefore the chip preparation time.