A potential new clinical analysis tool for sepsis diagnosis

micromotor-based immunoassay for on-the-move determination of sepsis biomarkers in blood samples from very low birth weight infants

A potential new clinical analysis tool for sepsis diagnosis

by Prof. A. Escarpa, Prof. M. A. López, Dr A. Molinero-Fernández, Dr M. Moreno-Guzmán and Dr L. Arruza Sepsis is a condition that can develop and become life threatening very quickly. The key to obtaining...

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by Prof. A. Escarpa, Prof. M. A. López, Dr A. Molinero-Fernández, Dr M. Moreno-Guzmán and Dr L. Arruza

Sepsis is a condition that can develop and become life threatening very quickly. The key to obtaining the best possible outcome is early diagnosis and quick treatment, which can be challenging when treating very low birth weight infants and limited volume blood samples. This article describes how micromotors, tiny particles that can propel themselves autonomously, can be adapted to achieve immunoassays for sepsis diagnosis in a very short time and with a very tiny sample volume.


Sepsis in general and particularly neonatal sepsis in the highly vulnerable population of very low birth weight infants (VLBWIs), is still a major cause of mortality and morbidity. Despite the significant advances in neonatal care and the increased understanding of the pathogenesis of the disease in recent years, the ability to intervene and modify the path of the disease has been only partially successful.

An early sepsis diagnosis and timely initiation of the therapy improves patient outcomes significantly. However, the diagnosis of sepsis is still one of the fundamental challenges in healthcare worldwide [1]. Early diagnosis is especially challenging in neonates owing to the low specificity and high variability of the symptoms, the lack of ideal sepsis biomarkers and the absence of optimal diagnostic tests [2]. Blood culture continues to be the gold standard method for diagnosing sepsis. However, its low sensitivity, the delay in obtaining results and the relatively large sample volume needed from VLBWIs, make it unsuitable for an early sepsis diagnostic. Likewise, the limitations in the available volume of blood in newborns can negatively affect the performance of the blood culture, because of the low rate of bacteremia in most cases.

As a result of the aspects cited above, new diagnostic tools are much desired by clinicians. These analytical methods should provide specificity, sensitivity, multiplexed analysis and fast results at the bedside. Moreover, they should also account for the limitations in blood volume availability in VLBWIs.

Micromotor-based immunoassays: a new clinical analysis tool

To help in solving these challenges, the use of catalytic self-propelled micromotors, adequately functionalized with relevant specific antibodies, is proposed as a new paradigm in the clinical assay scenario for the analysis of validated sepsis biomarkers, such as procalcitonin (PCT) and C-reactive protein (CRP). The pairing of the well-known features of immunoassays and the great potential of micromotors provides a synergistic combination to develop highly interesting point-of-care-testing (POCT) devices for sepsis diagnosis. A brief introduction to the nature of micromotors is described before further elaborating on this specific application.

Micromotors are microscopic-sized nanotechnological particles that have the ability to move autonomously, offering a plethora of new possibilities in clinical analysis and other fields [3]. Nature has provided many examples of its own machinery, such as kinesins, dynein, myosin or motor proteins to drive flagella and cilia in bacteria, sperm cells, etc. However, until the arrival of nanotechnology, the miniaturization of macroscopic objects was nothing more than a science fiction fantasy.

One of the main challenges of such micromachinery involves the power supply required for their propulsion. Although this energy can be provided by an external field or stimuli (ultrasound, magnetic and electrical field, light source, pH, temperature), the majority of micromotors developed so far harvest energy from the surrounding environment and are known as fuel-driven micromotors [4]. These devices are constructed with an inner layer that is composed of a catalytic material which triggers a chemical reaction upon interaction with another chemical substance present in the liquid environment (fuel). Among others, the most widely explored mechanism for creating movement is bubble propulsion. In this case, a favourable geometry is selected for the design of the micromotor, such as a tube with one closed end and one open end. Then, decomposition of the fuel micromotor-based immunoassay for on-the-move determination of sepsis biomarkers in blood samples from very low birth weight infants Sepsis Diagnosis by Prof. A. Escarpa, Prof. M. A. López, Dr A. Molinero-Fernández, Dr M. Moreno-Guzmán and Dr L. Arruza A potential new clinical analysis tool for sepsis diagnosis: September 2020 21 | (i.e. hydrogen peroxide) on the inner catalytic surface of the micromotor tube causes the formation of gaseous product (oxygen) that creates a stream of bubbles. The exiting of the stream of bubbles from the open end of the tube creates a strong thrust, propelling the micromotor in the opposite direction. Bubble-propelled micromotors can move with high speed of up to several millimetres per second [5].

However, micromotor construction can be customized to perform the desired application. In this case, a layer-by-layer architecture was easily electro-synthetized via a template-assisted method to provide the required functionalities [6]. Constructed in three layers, (Fig. 1) the external layer must contain carboxy moieties that can be further functionalized with the desired antibody to selectively recognize and capture the target protein and be able to successfully carry out all the immunoassay steps. The middle magnetic layer allows the guidance and stoppedflow operations of the self-propelled micromotors, providing easy manipulation/collection of the micromotor. The inner layer consists of the catalyst, which allows the bubble formation creating propulsion of the micromotor. The nanomaterials used for their construction can be chosen/selected accordingly to the desired properties [7].

Application of micromotors to sepsis diagnosis

With the knowledge of the techniques described above, our research group decided to apply this amazing technology to help solve the problem of sepsis diagnosis. Micromotor-based immunoassays have been used for the determination of both CRP and PCT levels, which are biomarkers relevant to sepsis diagnosis. Different nanomaterials have been used to construct the micromotors and different detection techniques have been tried, which demonstrates the versatility and potential of this technology.

Micromotor immunoassay detection of CRP

In a first approach, electrochemical detection of CRP using carbon-based micromotors was designed [8]. Self-propelled catalytic micromotors functionalized with anti-CRP specific antibodies were designed for capture and electrochemical detection of CRP using a sandwich format, and horseradish peroxidase (HRP)-labelled tracer. Micromotors with different carbon-based outer layer compositions were evaluated as active supports for antibody immobilization while maintaining propulsion efficiency. Among them, reduced graphene oxide (rGOx) resulted in the highest affinity and the best immunoassay performance. Platinum nanoparticles forming the inner layer catalysed the oxygen bubble generation by the decomposition of hydrogen peroxide on its surface.

Once the successful micromotors have been synthetized, the immunoassay can be developed. The number of rGOx micromotors, the immunoassay performance (antibody concentrations, non-specific adsorption incubation times), and propulsion conditions have to be studied in depth. In contrast with conventional immunosensors, where the analyte interacts with the usually immobilized specific antibody by diffusion or external stirring of the solution, self-propelled micromotors actively move around the sample to bind the analyte. Besides the expected efficient movement of the micromotors, the generated microbubble tails can enhance the fluid mixing, and consequently improve the efficiency of the biorecognition event. In this sense, the analysis time can be decreased significantly (<10|min) and extremely low sample volumes (7|μL) can be used, in which other stirring mechanisms are not effective enough.

These features are especially relevant when small volume samples are available, such as those from preterm babies with suspected sepsis.

Briefly, the CRP-micromotor immunoassay (MIm) procedure consists of the addition of 10|μL of micromotor suspension (around 2000 micromotors) modified with the capture antibody into a test tube. Then, the sample and detection antibody were added (10|μL total volume, of which 7|μL was the sample) to perform the sandwich immunocomplex in one step. In order to generate movement of the micromotors, the fuel was also added. This fuel mix contained surfactant to lower the interfacial tension as well as H2O2 as fuel, which allows the catalytic reaction responsible for the bubble formation in the inner layer of the micromotor. Under these conditions, the modified micromotors swim around the sample to find and capture the specific analyte for 5|min, boosting the biological interaction thanks to their autonomous movement in such a small volume. As a result of their magnetic characteristics (intermediate magnetic layer of nickel), the micromotors can be stopped and retained while the supernatant was removed. Once the immunoaffinity interactions were complete, electrochemical detection can be easily accomplished, re-suspending the micromotor–immunocomplexes in just 45|μL of hydroquinone solution (electrochemical mediator) and magnetically captured onto the surface of a portable and disposable screen-printed carbon electrode. Amperometric measurements were performed after the addition of hydrogen peroxide solution as enzymatic substrate.

Under these circumstances, the obtained analytical characteristics are highly competitive with other CRP immunoassays reported in the literature [8]. A linear working range from 2 to 100|μg/mL, (r|=|0.992), limit of detection (LOD) and limit of quantification (LOQ) of 0.8|μg/mL and 2.0|μg/mL, respectively, were obtained. Inter-assay precision for two different concentration values was 8%, whereas inter-assay precision was lower than 15% (n|=|5 days) for micromotors produced in different batches.

In order to evaluate the applicability in neonate clinical samples, the propulsion of the micromotors was tested in serum and plasma media, to compare with their behaviour in buffer. Although a diminished speed was observed in serum and plasma, it did not affect their function and indeed propulsion was improved in plasma by adjusting the fuel concentration. Hence, serum certified reference material (SCRM) and unique plasma samples from neonates with suspected sepsis were analysed.

The achieved results revealed an excellent accuracy of our MIm (Er|=|1%). Moreover, the results obtained by our MIm in comparison with those reported by the Hospital laboratory using standard analytical methods (BRAHMS CRPus KRYPTOR) for preterm neonate plasma samples, did not show significant differences (P<0.05), allowing the analysis of clinically relevant samples.

Seeking to fulfil all the requirements for performing on-site/bedside assays, the micromotor-based immunoassay for CRP described above was fully integrated into a microfluidic platform [9]. In this new approach, both main immunoassays steps, immunocomplex formation and detection steps, were performed in the same microfluidic system, in a fully automated way, and so dispensing with the need for human handling. This on-chip MIm approach blends the advantages of the micromotors with those of microfluidic technology, giving place to a new alternative for sepsis POCT development. Among other advantages, this CRP assay is fast (<10|min), easy-to-use, automated, requires low sample volume (7|μL) and presents an improvement of an analytical characteristic (LOD|=|0.54|μg/mL). Furthermore, the good results obtained after SCRM (Er<10%) and the analysis of neonate samples demonstrated its applicability in a real clinical environment.

MIm detection of PCT

As previously stated, the flexibility of the technology allowed changing the target analyte by simply modifying the specific antibody bound to the outer layer of the micromotors, and the detection technique using an adequate label. In this sense, the second approach deals with a micromotor-based fluorescence immunoassay for PCT determination in samples from neonates [10]. This time, different polymeric outer layers were tested, with COOH-polypyrrol being the polymer producing a higher degree of antibody functionalization and improving the sensitivity of the assay. The operating mode is similar to the previously developed CRP-MIm. However, in this case, detection is carried out on a fluorescence microscope by directly positioning 1|μL of the previously formed micromotor–immunocomplexes onto a microscope slide to perform the fluorescence measurements at excitation and emission wavelengths of 490|nm and 520|nm, respectively. These fluorescence signals were recorded and analysed by the high-resolution camera and its software associated with the microscope. A wide working linear range between 0.50 and 150|ng/mL, and LOD and LOQ of 0.07 and 0.50|ng/mL were obtained, respectively. Precision, even for intra- or inter-assay was lower than 9%. The applicability to real clinical samples was evaluated by analysing plasma samples from very low birth weight neonates. The results obtained by our MIm did not show significant differences with the PCT levels declared by the Hospital (BRAHMS PCT) (P<0.05).


In summary, these new micromotor-based immunoassay approaches exhibited key advantages such as simplicity, rapidity, miniaturization, automatization potential and reliability of analysis, using extremely low sample volumes and covering the entire range of concentrations involved in the clinical sepsis diagnosis (Fig. 2). Therefore, MIms are presented as future tools for early diagnostics, which is essential for timely treatment and the adequate guidance of antibiotic therapy as well as in the development of POCT devices.

The authors

Alberto Escarpa*1,2 , Miguel Ángel López 1,2 , Águeda Molinero-Fernández 1
PhD, María Moreno-Guzmán 3 and Luis Arruza 4
1Department of Analytical Chemistry, Physical Chemistry and Chemical
Engineering, University of Alcalá, Alcalá de Henares, E-28871 Madrid, Spain
2Chemical Research Institute Andres M. del Rio, University of Alcalá,
E-28871 Madrid, Spain
3Department of Chemistry in Pharmaceutical Sciences, Analytical Chemistry,
Faculty of Pharmacy, Universidad Complutense de Madrid, E-28040 Madrid,
4Division of Neonatology, Instituto del Niño y del Adolescente, Hospital
Clinico San Carlos – IdISSC, E-28040 Madrid, Spain

*Corresponding author
E-mail: alberto.escarpa@uah.es

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