[en] This work describes a method based on an enzymatic assay to measure the free concentration of piperacillin (a [Formula: see text]-lactam antibiotics) in blood serum. The assay is based on the release of fluorescent umbelliferone, upon hydrolysis by BlaP99 [Formula: see text]-lactamase of a substrate in competition with piperacillin. The assay is implemented in a lab-on-a-disk (LoaD) setup, in which samples and reagents are manipulated with robust centrifugal microfluidic techniques. To encounter the needs and constraints of Therapeutic Drug Monitoring (TDM) at bedside, the system is entirely automated and miniaturized. Within a few minutes and from a few microliters of serum, it provides piperacillin concentration measurements in a clinically meaningful range. This paper describes the design and characterization of the chemical kinetics and the microfluidic strategy underlying this assay. The data are compared to measurements made in well-plates with a conventional method.
Disciplines :
Biotechnology Mechanical engineering
Author, co-author :
Verlaine, Olivier ; Université de Liège - ULiège > Département des sciences de la vie > Centre d'Ingénierie des Protéines (CIP)
Amoroso, Ana Maria ; Université de Liège - ULiège > Département des sciences de la vie > Génétique et physiologie des microalgues
Remy, Maxence; Institute of Condensed Matter and Nanosciences, Université Catholique de Louvain, Place Louis Pasteur 1, bte L4.01.06, 1348, Louvain-la-Neuve, Belgium
Wautier, Stéphanie; Institute of Condensed Matter and Nanosciences, Université Catholique de Louvain, Place Louis Pasteur 1, bte L4.01.06, 1348, Louvain-la-Neuve, Belgium
Robiette, Raphaël; Institute of Condensed Matter and Nanosciences, Université Catholique de Louvain, Place Louis Pasteur 1, bte L4.01.06, 1348, Louvain-la-Neuve, Belgium
Joris, Bernard ; Université de Liège - ULiège > Département des sciences de la vie
Gilet, Tristan ; Université de Liège - ULiège > Département d'aérospatiale et mécanique > Microfluidique
Language :
English
Title :
Enzymatic assay in a lab-on-a-disk format to measure free piperacillin concentration in serum.
Publication date :
17 March 2026
Journal title :
Biomedical Microdevices
ISSN :
1387-2176
eISSN :
1572-8781
Publisher :
Springer Science and Business Media LLC, United States
Abdul-Aziz MH Alffenaar JC Bassetti M Bracht H Dimopoulos G Marriott D Neely MN Paiva JA Pea F Sjovall F Timsit JF Udy AA Wicha SG Zeitlinger M De Waele JJ Roberts JA Infection section of european society of intensive care medicine (esicm); pharmacokinetic/pharmacodynamic and critically ill patient study groups of european society of clinical microbiology and infectious diseases (escmid); infectious diseases group of international asso-ciation of therapeutic drug monitoring and clinical toxicology (iatdmct); infections in the icu and sepsis working group of international society of antimicrobial chemotherapy (isac). Antimicrobial therapeutic drug monitoring in critically ill adult patients: a position paper Intensive Care Med. 2020 46 6 1127 1153 10.1007/s00134-020-06050-1
Abdul-Aziz MH Brady KM Cotta MO Roberts JA Therapeutic drug monitoring of antibiotics: Defining the therapeutic range Ther. Drug Monit. 2022 44 1 19 31 10.1097/FTD.0000000000000940
Abdul-Aziz MH Hammond NE Brett SJ Cotta MO De Waele JJ Devaux A Di Tanna GL Dulhunty JM Elkady H Eriksson L Hasan MS Khan AB Lipman J Liu X Monti G Myburgh J Novy E Omar S Rajbhandari D Roger C Sjövall F Zaghi I Zangrillo A Delaney A Roberts JA Prolonged vs intermittent infusions of -lactam antibiotics in adults with sepsis or septic shock: A systematic review and meta-analysis JAMA 2024 332 8 638 649 10.1001/jama.2024.9803
Berkel SS Brem J Rydzik AM Salimraj R Cain R Verma A Owens RJ Fishwick CWG Spencer J Schofield CJ Assay platform for clinically relevant metallo--lactamases J. Med. Chem. 2013 56 17 6945 6953 10.1021/jm400769b
A. Brans, B. Joris, M. Delmarcelle, J. Marchand, C. Hammaecher, P. Tulkens, E. Goormaghtigh, J. De Coninck, Enzymatic method for rapid and continuous determination of beta-lactamic antibiotics. WO2013053953, WO2013053953
Bush K Bradford PA -lactams and -lactamase inhibitors: An overview Cold Spring Harbor Perspect. Med. 2016 6 8 10.1101/cshperspect.a025247 025247
Bush K Sykes RB Methodology for the study of -lactamases Antimicrob. Agents Chemother. 1986 30 1 6 10 10.1128/aac.30.1.6
Dhaese S Van Vooren S Boelens J De Waele J Therapeutic drug monitoring of -lactam antibiotics in the icu Expert Rev. Anti-infect. Ther. 2020 18 11 1155 1164 10.1080/14787210.2020.1788387
Ducrée J Haeberle S Lutz S Pausch S Stetten F Zengerle R The centrifugal microfluidic bio-disk platform J. Micromech. Microeng. 2007 17 103 115 10.1088/0960-1317/17/7/S07
Duffy DC Gillis HL Lin J Sheppard NF Kellogg GJ Microfabricated centrifugal microfluidic systems: characterization and multiple enzymatic assays Anal. Chem. 1999 71 20 4669 4678 10.1021/ac990682c
Galleni M Lindberg F Normark S Cole S Honoré N Joris B Frère J-M Sequence and comparative analysis of three enterobacter cloacae ampc -lactamase genes and their products Biochem. J. 1988 250 753 760 10.1042/bj2500753
Gao W Xing B Tsien RY Rao J Novel fluorogenic substrates for imaging beta-lactamase gene expression J. Am. Chem. Soc. 2003 125 37 11146 11147 10.1021/ja036126o
Gholizadeh A Mazzucchelli G Gilet T Flipping: A valve-free strategy to control fluid flow in centrifugal microfluidic systems Sens. Actuators B-Chem. 2024 412 10.1016/j.snb.2024.135778 135778
R. Gorkin, J. Park, J. Siegrist, M. Amasia, B.S. Lee, J.M. Park, J. Kim, H. Kim, M. Madou, Y.K. Cho, Centrifugal microfluidics for biomedical applications. Lab Chip 10(14), 1758–1773 (2010). https://doi.org/10.1039/b924109d
Haeberle S Brenner T Zengerle R Ducrée J Centrifugal extraction of plasma from whole blood on a rotating disk Lab Chip 2006 6 776 781 10.1039/b604145k
B. Joris, A. Amoroso, O. Verlaine, Methods for determining the concentration of a carbapenem antibiotic in a biological sample. EP3997240, EP3997240
D.J. Kinahan, K. McConville, B. Henderson, M. McCaul, E. McNamara, D. Diamond, J. Ducrée, Digital pulse actuated flow control on a centrifugal disc towards multiparameter water quality monitoring. In: 20th International conference on miniaturized systems for chemistry and life sciences (2016)
Kuo J-N Li B-S Lab-on-cd microfluidic platform for rapid separation and mixing of plasma from whole blood Biomed. Microdevices 2014 16 549 558 10.1007/s10544-014-9857-1
Mabilat C Gros MF Nicolau D Mouton JW Textoris J Roberts JA Cotta MO Belkum A Caniaux I Diagnostic and medical needs for therapeutic drug monitoring of antibiotics Eur. J. Clin. Microbiol. Infect. Dis. 2020 39 5 791 797 10.1007/s10096-019-03769-8
Peshin S Madou M Kulinsky L Microvalves for applications in centrifugal microfluidics Sensors 2022 22 22 8955 10.3390/s22228955
Power P Galleni M Ayala JA Gutkind G Biochemical and molecular characterization of three new variants of ampc -lactamases from morganella morganii Antimicrob. Agents Chemother. 2006 50 3 962 967 10.1128/aac.50.3.962-967.2006
Roberts JA Abdul-Aziz MH Lipman J Mouton JW Vinks AA Felton TW Hope WW Farkas A Neely MN Schentag JJ Drusano G Frey OR Theuretzbacher U Kuti JL International society of anti-infective pharmacology and the pharmacokinetics and pharmacodynamics study group of the european society of clinical microbiology and infectious diseases. Individualized antibiotic dosing for patients who are critically ill: challenges and potential solutions Lancet Infect Dis 2014 14 6 498 509 10.1016/S1473-3099(14)70036-2
Strohmeier O Keller M Schwemmer F Zehnle S Mark D Stetten F Zengerle R Paust N Centrifugal microfluidic platforms: advanced unit operations and applications Chem. Soc. Rev. 2015 44 17 6187 6229 10.1039/c4cs00371c
Tilanus A Drusano G Optimizing the use of beta-lactam antibiotics in clinical practice: A test of time Open Forum Infect. Dis. 2023 10 7 305 10.1093/ofid/ofad305
Wicha SG Märtson AG Nielsen EI Koch BCP Friberg LE Alffenaar JW Minichmayr IK International society of anti-infective pharmacology (isap), the pk/pd study group of the european society of clinical microbiology, infectious diseases (epasg). From therapeutic drug monitoring to model-informed precision dosing for antibiotics Clin. Pharmacol. Ther. 2021 109 4 928 941 10.1002/cpt.2202.7
Wong G Briscoe S Adnan S McWhinney B Ungerer J Lipman J Roberts JA Protein binding of -lactam antibiotics in critically ill patients: Can we successfully predict unbound concentrations?> Antimicrob. Agents Chemother. 2013 57 12 6165 6170 10.1128/aac.00951-13
Wouters J Charlier P Monnaie D Frère JM Fonzé E Expression, purification, crystallization and preliminary x-ray analysis of the native class c beta-lactamase from enterobacter cloacae 908r and two mutants Acta Crystallogr. D 2001 57 1 162 164 10.1107/s0907444900016413
Wu W Zhao Q Cai G Zhang B Suo Y Liu Y Jin W Mu Y All-in-one escherichia coli viability assay for multi-dimensional detection of uncomplicated urinary tract infections Anal. Chem. 2022 94 51 17853 17860 10.1021/acs.analchem.2c03604
Wu W Cai G Liu Y Suo Y Zhang B Jin W Yu Y Mu Y Direct single-cell antimicrobial susceptibility testing of escherichia coli in urine using a ready-to-use 3d microwell array chip Lab Chip 2023 23 10 2399 2410 10.1039/D2LC01095J
