On-the-fly rapid immunoassay for neonatal sepsis diagnosis: C-reactive protein accurate determination using magnetic graphene-based micromotors
Introduction
Micro and nanomotors that convert chemical energy or an external stimulus (magnetic, ultrasound or light) into autonomous propulsion have gained a great and continuous growing attention in the last decade (Zha et al., 2018; Karshalev et al., 2018). Among other fields, clinical diagnostic and biosensing capabilities of these micro/nano synthetic machines have opened a plethora of new bioanalytical applications. Some of the most important challenges in biomedical applications are the low detection limits required for early disease detection and the ultra-small sample volumes commonly available. In these situations, sensitivity is usually limited by the inefficient transport of the analyte towards the bioreceptor for their interaction, due to the quiescent conditions ordinarily used in such assays or the ineffective convection transport in so low volume samples. In this sense, the self-propelled and autonomous propulsion of bioreceptor-modified micromotors around a complex sample, to find and bound the analyte, represents a new paradigm in analytical chemistry (Wang, 2016; Kong et al., 2018). In particular, bubble-propelled catalytic micro/nano motors are especially appropriate due to their rapid propulsion and the mixing associated effect by the generated microbubbles tail. This fluid mixing effect leads to a favourable hydrodynamic environment, which enhances the kinetics of the binding reaction and promotes the interaction of the target analytes with the receptor (Wang, 2016; Orozco et al., 2014; Morales-Narváez et al., 2014; Campuzano et al., 2011a; Kagan et al., 2011). This process is especially efficient in ultraminiaturized samples with minimal sample preparations.
Different biological receptors such as nucleic acids (Kagan et al., 2011), aptamers (Esteban-Fernández de Ávila et al., 2016; Molinero-Fernández et al., 2017; Molinero-Fernández et al., 2018), and lectins (Campuzano et al., 2011b; Maria-Hormigos et al., 2017) have been immobilized on the surface of micromotors to perform new “on-the-fly” detection schemes with higher efficiency and shorter times. Specially, antibodies-modified micromotors have demonstrated their ability to detect different targets molecules (García et al., 2013; Vilela et al., 2014) such as tumoral cells (Balasubramanian et al., 2011), cancer biomarkers (Yu et al., 2014), spores of Bacillus anthracis (Orozco et al., 2015) and cortisol (Esteban-Fernández de Ávila et al., 2017).
The material used for the construction of the outer micromotor layer is crucial, since it affects its capacity of binding biomolecules on its surface as well as micromotors' propulsion efficiency. Different nanomaterials and strategies have been previously explored for the construction of the outer layer supporting the biomolecules such as gold sputtering that produces only a partial modification of the half outer micromotors layer, affecting their overall capacity of biomolecule immobilization and mobility (Esteban-Fernández de Ávila et al., 2017); gold nanoparticles decoration that implies additional and more complex stages (Yu et al., 2014); or electro-copolimerization (García et al., 2013), which need rigorous control of monomers concentration and doping in order to maintain the morphology and performance of the micromotors.
On the other hand, carbon nanomaterials possess unique physical and chemical properties that allow both, biomolecules immobilization in the micromotor outer layer (Maria-Hormigos et al., 2018) as well as high micromotor speeds when suitable catalysts are also used in the inner layer (Martín et al., 2015; Maria-Hormigos et al., 2016).
On the other hand, C-reactive protein (CRP) is a homopentameric plasmatic protein with a molecular weight of 118 KDa that is produced mainly in liver and lung as response to inflammation (Vashist et al., 2016). CRP is a widely studied protein due to its high interest as early indicator of infectious or inflammatory conditions, related to various diseases, disorders and pathological conditions (Agrawal et al., 2014; Brito et al., 2015; Clyne y Olshaker, 1999). An elevation in its plasmatic levels can be directly related to the severity of the disease, increasing up to 1000-fold during an acute inflammation phase (Hisamuddin et al., 2015; Yuan et al., 2013; Boonkasidecha et al., 2013). Elevated CRP levels have been directly related to the risk of suffering cardiovascular disease, where blood serum concentration ranges <1, 1–3 and >3 μg/mL correlates with low, moderate and high risk, respectively. Especially important is the role of CRP in the diagnosis of sepsis. Early and accurate diagnosis is crucial to decrease mortality and to avoid unnecessary exposure to antibiotics, in order to reduce antibiotic resistance and severe side effects in patients. In this sense, CRP has demonstrated to be a late biomarker of neonatal infections and a good marker of the response to antibiotic therapy (Simon et al., 2004; Vashist et al., 2016). Values higher than 10 μg/mL in blood, can be considered positive for sepsis.
However, sepsis diagnosis is still a challenge and complex phenomenon due to the unspecific signs and symptoms and the absence of an ideal biomarker. Besides, the need to carry out serial analysis to improve diagnosis and disease monitoring, constitutes an additional problem, especially for those patients where low volume samples are available such as neonates. Hence, the development of reliable, cost-effective, sensitive, and fast approaches for CRP detection and monitoring, which in turn allow the use of low sample volumes, is particularly important in clinical analysis.
CRP determination has been carried out by different techniques such as chemiluminescence and fluorescence immunoassays, or enzyme-linked immunosorbent and immunoturbidimetric assays. They are sensitive, but usually suffer from important sample and reagent consumption. Moreover, routinely, they are performed in central laboratories and results are delayed, which is highly incompatible with the need to make quick decisions. In that sense, new approaches to surpass those limitations have been developed during the last years (Vashist et al., 2016). Among them, electrochemical immunosensors are one of the most attractive techniques for the detection of clinical samples by virtue of their low cost, high sensitivity, rapidity, and portability. (Yáñez-Sedeño et al., 2017). Several electrochemical immunosensors for CRP have been developed (Vermeeren et al., 2011; Esteban-Fernández de Ávila et al., 2013; Kumar and Prasad, 2012; Ibupoto et al., 2012; Bryan et al., 2012; Fakanya and Tothill, 2014; Yagati et al., 2016; Liu et al., 2016; Thangamuthu et al., 2018; Kowalczyk et al., 2018; Boonkaew et al., 2019; Molinero-Fernández et al., 2019). However, to the best of our knowledge, the micromotors technology has not been explored for CRP determination.
Because of the use of micromotors as new tools in immunoassays provides a new paradigm, as stated above, our hypothesis is that antibody-functionalized micromotors can swim autonomously around the sample to bind actively the specific analyte, without the need of external stirring. The expected efficient movement of the micromotors and the generated microbubbles tails, can enhance the fluid mixing, and consequently improving the efficiency of the biorecognition event. This principle can favours a fast strategy for CRP determination in extremely small volume samples, such as those obtained from preterm babies with sepsis suspicion.
In this work, we have proposed the first micromotor-based immunoassay (MIm) for the on-the-fly CRP determination. In the following sections, we will demonstrate the analytical potency of this biosensing approach for CRP rapid and accurate determination in clinical samples such as preterm infants’ plasma.
Section snippets
Reagents and solutions
CRP (8C72) and two paired monoclonal mouse anti-human CRP antibodies (4C28C HRP-conjugated and 4C28B biotinylated) were obtained from HyTest (Turku, Finland).
Dilution of CRP and anti-human CRP antibodies were prepared in PBS, 0.1M phosphate (Scharlau, 99%), 0.138M NaCl (Scharlau, 99%) and 2.7 mM KCl (Scharlau, 99%) buffer solution pH 7.5.
Enzyme substrate PERDROGEN™ 30% H2O2, Hydroquinone, Bovine serum albumin (BSA), Streptavidin, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC),
Micromotor-based immunoassay strategy
Tubular micromotors constituted by rGO as functionalization support outer layer covered with streptavidin, an intermediate magnetic Ni and internal PtNPs catalytic layer were electrosynthetized for the on-the-fly immunoassay strategy. The micromotor immunoassay design is depicted in Fig. 1. Streptavidin rGO/Ni/PtNPs micromotors were previously functionalized with anti-CRP capture antibody. These functionalized micromotors were left in a solution that contained the protein target (CRP), anti-CRP
Conclusions
Analytical capabilities of smartly designed and constructed rGO/Ni/PtNPs micromotors into three layers (rGO for antibody sandwich functionalization, Ni for magnetic guidance and stopped flow operations and PtNPs for catalytic bubble propulsion) for CRP immunosensing on the fly has been demonstrated to be very useful for neonatal sepsis diagnosis.
Under controlled propulsion conditions, the new approach using magnetic anti-CRP rGO/Ni/PtNPs micromotors offered a rapid on-the-fly (5 min) and
CRediT authorship contribution statement
Águeda Molinero-Fernández: Investigation, Formal analysis, Writing - original draft, Visualization, Conceptualization, Methodology. Luis Arruza: Resources, Writing - review & editing. Miguel Ángel López: Conceptualization, Methodology, Supervision, Writing - review & editing. Alberto Escarpa: Resources, Writing - review & editing, Supervision, Project administration, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness (CTQ2017-86441-C2-1-R), the TRANSNANOAVANSENS program (S2018/NMT-4349) from the Community of Madrid and La Caixa Impulse program (CI017-00038) (A.E and MA.L.G ).
A.M.F. acknowledges the FPU fellowship from the Spanish Ministry of Education, Culture and Sports.
We thank Jose Miguel González Dominguez from the ICB-CSIC, Álvaro Colina from the University of Burgos and Juan Víctor Perales from the University of
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