Detection of canine and equine procalcitonin for sepsis diagnosis in veterinary clinic by the development of novel MIP-based SPR biosensors

Highlights

• Methods for procalcitonin detection are poorly investigated in veterinary medicine.

• The only analytical method available is represented by immunoassays.

• Molecular imprinting is a widely explored strategy to develop biosensors platforms.

• Here we report two MIP receptors for canine and equine procalcitonin.

• PDA- and PNE-based MIPs are optimized and compared by SPR.

Abstract

Procalcitonin (PCT) has emerged as a promising biomarker for the rapid identification of sepsis both in human and veterinary medicine. Nevertheless, the only analytical method currently available for the detection of PCT in veterinary species, is represented by immunoassays, useful only for research purposes. In this work, we report the development of two biosensors which utilize molecularly imprinted polymers (MIPs) for the detection of canine and equine PCT. Dopamine (DA) and norepinephrine (NE) were used as monomers for the synthesis of the MIP films on surface plasmon resonance (SPR) gold chips and the imprinting efficiency of canine and equine PCT in terms of binding affinity toward the analyte, selectivity, and sensitivity were compared. After optimization in buffer conditions, PCTs calibration was successfully achieved also in animal plasma, with good specificity and reproducibility. More effective protein binding and imprinting was obtained with polynorepinephrine (PNE) for both PCTs, and the SPR biosensors were able to detect the biomarkers in plasma with a LOD of 15 ng mL⁻¹ and 30 ng mL⁻¹ respectively for equine and canine PCT.

Introduction

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection and represents the leading cause of morbidity and mortality from infection in humans as well as in veterinary patients, especially if not diagnosed and treated promptly [1,2]. The early diagnosis of sepsis and the evaluation of its severity are fundamental to prevent the onset of a septic shock and to increase the survival rate. Among the sepsis biomarkers (e.g. transcription factors, lactate, interleukins, C-reactive protein), PCT resulted to be a more specific indicator of bacterial infection or severity of infection, and to be a good control of the success of a therapeutic procedure [[3], [4], [5]]. The PCT level in serum of healthy donors is commonly below 0.05 ng mL⁻¹, while it quickly increases up to 1.000 folds in serum from patients with sepsis, i.e. within 6–12 h [6]. While PCT immuno-based detection as sepsis predictor is assessed in humans, using reference commercial platforms (BRAHMS®, Vidas Biomerieux, etc.) [7], in veterinary patients there is a lack of validated assays [2,8] and, therefore, of clinical studies. The few available are mostly limited to dogs and horses. Moreover, in the last two decades several immuno-based Point of Care Tests have become available for human PCT detection, exclusively based on anti-calcitonin and anti-katacalcin antibodies, employing different detection strategies, i.e. type of signal recorded, its amplification strategy, and the platform adopted [[9], [10], [11], [12]]. In contrast to the high interest for labeled PCT immunoassays, in literature there are very few papers dedicated to label-free biosensors for PCT detection, and they all refer to human PCT with ng mL⁻¹ detection levels and are mainly based on electrochemical transduction; Lim et al. (2017) synthesized as capturing agents, i.e. receptors, a series of synthetic peptides modified with cysteine residues at the C-terminus to facilitate thiol self-assembly, reporting a binding constant (K_d) of 0.39 ± 0.11 nM and limit of detection of 12.5 ng mL⁻¹ for procalcitonin in PBS in the case of the BP3 peptide [13]. Electrochemical immunosensors are also reported, eventually using nanomaterials to achieve sensitive PCT detection. Li et al. (2015) described an immunosensor displaying a detection limit of 6 pg mL⁻¹ for PCT using a new signal-amplification strategy based on a nanocomposite derived from amino-functionalized C60 nanoparticles, ferrocene carboxylic acid and platinum nanoparticles (PtNPs) [14]. Gao et al. (2020) developed a label-free electrochemical immunosensor using toluidine blue functionalized NiFe Prussian-blue analog nanocubes (NiFe PBA nanocubes@TB) as a signal amplifier. The developed immunosensor exhibited favorable performance for PCT detection with a linear range from 0.001 to 25 ng mL⁻¹ and a detection limit of 3 × 10⁻⁴ ng mL⁻¹ [15].

Beside electrochemical transduction, only one example of optical sensing is available in literature based on the use of biomimetic receptors. Sener et al. (2013) developed a molecularly imprinted polymer for the SPR-based detection of hPCT by using as functional monomers a solution of hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EGDMA). As reported, the sensor accounted for a limit of detection of 9.9 ng mL⁻¹ in both phosphate buffer and simulated blood plasma [16]. Conversely, in veterinary patients, only three commercial ELISA kits are available for dogs or horses, not fully validated, and suitable only for research purposes. In particular, only one ELISA-type immunoassay is reported in literature for equine PCT, based on human antibodies since there is no availability of specific antibodies [[17], [18], [19], [20], [21]].

In this framework, we considered the need to develop innovative biomimetic receptors to overcome limitations of current antibodies, i.e. availability, specificity, cost and stability. Molecularly Imprinted Polymers (MIPs) technology coupled to biosensing is particularly interesting thanks to its low-cost and long-term stability features [22]. In particular, macromolecules imprinting has received recently a growing interest despite the large size and the structural complexity of proteins (large number of functional groups) impeding molecules to penetrate the polymer network with consequences as nonspecific interactions, poor selectivity, and cross reactivity [23]. The choice of functional monomers defines the porous structure and the surface area of the MIP and plays an important role in the creation of binding sites [[24], [25], [26]]. Methacrylic acid and derivatives have been commonly used as functional monomers due to their structure related to polyethylene glycol (PEG), and to their characteristic being capable to act as a hydrogen-bond donor and acceptor [16,25,[27], [28], [29]]. Despite some good results, these monomers are not really suitable for biomolecules imprinting, presenting some limitations as the necessity of harsh conditions for polymerization and template removal, polymer hardness, and limited mechanical strength of such materials [30]. To overcome these drawbacks, recent research for functional monomers, that could positively influence the properties of the final polymer, led to a new generation of biopolymer-based MIPs. In this field, the neurotransmitters dopamine (DA) and, more recently, its structural analogous norepinephrine (NE) have emerged as very promising monomers for biomolecules imprinting. Due to the redox potential of the catechol moiety, the two catecholamines share the same ability to self-polymerize and to form adherent films on almost any kind of surface [31]. DA has already displayed excellent performances as a functional monomer for molecular imprinting technique, for the recognition and separation of small drugs, proteins, nucleic acids, and microorganisms [[32], [33], [34], [35], [36]]. Contrary to the impressive success of DA in molecular imprinting, its derivative NE, that differs from DA just for an additional hydroxyl group, has only recently been investigated as a new functional monomer for MIPs synthesis in biosensing [37]. In particular, Baldoneschi et al. reported on a polynorepinephrine (PNE)-based biosensor for the detection of troponin I, a crucial biomarker of acute myocardial infarction, coupled to SPR detection, highlighting the reduced non-specific adsorption of proteins on PNE with respect to polydopamine (PDA) on chip sensor surface [38]. Here, PNE and PDA were used and compared for the first time to produce MIPs able to bind and quantify canine and equine PCT. The MIPs have been characterized in terms of binding affinity toward the analyte, specificity, and sensitivity using real-time and label-free surface plasmon resonance (SPR) biosensing. Finally, the MIPs-based biosensors were tested on real canine and equine sera samples to evaluate its potential application for sepsis diagnosis in veterinary analysis.