Introduction
(Article introduction authored by ICU Editorial Team)
Improving sepsis diagnosis in newborns, given their extended life expectancy, is paramount, yet early detection remains a significant challenge. However, a breakthrough emerges with the development of a micromotor-based dual aptassay.
By concurrently detecting biomarkers like procalcitonin (PCT) and interleukin-6 (IL-6), this innovative approach enhances diagnostic precision, crucial for timely intervention.
Notably, this method requires just a minute sample and delivers results within a mere 15 minutes, revolutionizing the landscape of neonatal sepsis diagnosis.
Its remarkable sensitivity and correlation with established hospital methods underscore its reliability, making it a promising tool for identifying sepsis in high-risk newborns promptly.
This micromotor technology heralds a new era in low sample volume-based diagnostics, offering a paradigm shift in neonatal care. Its ability to provide expeditious and accurate results with minimal sample requirements makes it a game-changer in diagnosing neonatal sepsis.
By facilitating swift and informed decisions about antibiotic therapy initiation, this approach empowers clinicians to intervene effectively in the critical early stages of sepsis, potentially saving lives and improving outcomes for vulnerable newborns
Experimental
The specific aptamers for PCT (5′-GCG GAT GAA GAC TGG TGT GTG GGG GAG GGG TGA GTT TTA GTG TTT TTG TTG GTT GGC GGC CCT AAA TAC GAG CAA C-3′) and IL-6 (5 ́-GTT GCT CGT ATT TAG GGC CGA GGT ACG AGT GTC TTT GGG ATC TGC ATT CTC CTG CGT GAC ACC AGT CTT CAT CCG C-3′) for procalcitonin (PCT) and interleukin-6 (IL-6) were synthesized using SELEX technology.
The aptamer sequences were identified through multiple selection rounds using recombinant forms of the respective proteins immobilized on Ni–NTA Agarose resin.
After enrichment analysis via real-time PCR, massive sequencing (NGS—Illumina) was conducted to identify the aptamer sequences against IL-6 and PCT.
The aptamers were synthesized with fluorescent labels (Alexa488 6-FAM for IL-6 and Alexa405 for PCT) to facilitate the dual aptassay procedure.
Dilutions of PCT and IL-6 were prepared in phosphate-buffered saline (PBS) solution, while the aptamers were prepared in phosphate-buffered-MgCl2 solution.
Additionally, various chemicals and materials, including graphene oxide, sulfuric acid, nickel salts, platinum salts, and polycarbonate membranes, were utilized in the experimental setup.
All chemicals used were of analytical grade, and deionized water was obtained from a purification system.
Samples
The study recruited very preterm infants (<32 weeks gestational age) with very low birth weight (<1000 g) admitted to the neonatal intensive care unit (NICU) due to clinical suspicion of late-onset sepsis (>72 h of life).
Symptoms indicating infection included cardiorespiratory instability, apnea, thermal dysregulation, neurological deterioration, or general ill appearance in previously stable infants.
Parental informed consent was obtained before inclusion, and blood samples were collected as part of routine sepsis evaluation, including tests for acute phase reactants levels (C-reactive protein, procalcitonin, and IL-6), white cell count, and blood cultures.
Additional blood samples for research purposes were collected only after informed consent, ensuring no interference with patient management without parental permission. The study was approved by the local Ethics Committee and the University of Alcalá. Blood samples were processed in the hospital laboratory for IL-6 and PCT measurement using chemiluminescent immunoassay and immunofluorescence, respectively.
Another aliquot of blood was centrifuged for serum separation and transported to the research laboratory for further analysis.
Apparatus
The micromotor (MM) electrosynthesis was conducted using a μ-Autolab Type III apparatus. For immunoassay incubation and magnetic MM handling, an Advanced VortexMixer-ZX3 from VWR, Thermosaker TS-100C from Biosan, and the Magnetic block DynaMag-2 from ThermoFisher were utilized.
Scanning electron microscopy (SEM) images were acquired with a JEOL JSM 6335F, and X-ray analysis was performed using an EDX detector coupled to an SEM instrument. Fluorescence measurements were conducted using an inverted optical microscope (Nikon Eclipse 80i) equipped with a Hamamatsu digital camera C11440, specific objectives and filters for PCT (λex, 377 nm; λem, 447 nm) and IL-6 (λex, 470 nm; λem, 520 nm) detection. Image and video capture were facilitated by the NIS Elements AR 3.2 software.
Electrosynthesis of micromotors
Tubular graphene oxide (GO)/Ni/platinum nanoparticles (PtNPs) micromotors (MM) were synthesized through electrodeposition of three distinct functional layers: an inner catalytic layer of PtNPs for bubble propulsion, an intermediate magnetic layer of Ni for magnetic handling, and an outer sensing layer of GO. The membrane, treated with a sputtered thin gold film on the S4-branched side, served as the working electrode during synthesis.
The electrodeposition process involved three steps: (i) cyclic voltammetry (CV) deposition of the GO outer layer, (ii) galvanostatic deposition of the Ni intermediate layer, and (iii) amperometric deposition of the Pt inner layer.
After deposition, the gold layer was polished to remove excess material, and the membrane was thoroughly washed with CH2Cl2, isopropanol, ethanol, and ultrapure water. The resulting MM, stored in ultrapure water at 4°C, exhibited reproducible characteristics in shape, size, and motility, demonstrating the effectiveness of the synthesis method.
MM dual aptassay procedure
The micromotor (MM) suspension was incubated with specific aptamers against procalcitonin (PCT) or interleukin-6 (IL-6) to obtain quenched MM-AptPCT or MM-AptIL-6, respectively.
After incubation and washing to remove free aptamers, a mixture of MM-AptPCT and MM-AptIL-6, along with PCT and IL-6 standards or samples, was prepared in BSA solution and hydrogen peroxide (H2O2) as a propulsion reagent.
This solution was deposited into an ELISA microwell for simultaneous recognition of both proteins by the autonomous movement of MM.
The solution was then analyzed using an inverted optical microscope equipped with fluorescence filters specific for PCT and IL-6.
Fluorescence signals were analyzed using software associated with the microscope and fitted with an equation to determine the analyte concentration.
Limit of detection (LOD) and limit of quantification (LOQ) were calculated based on standard deviation and slope obtained from calibration curves of PCT and IL-6 fluorescence signals.
Results and discussion
MM-based dual aptassay: the strategy
Figure 1 illustrates a scheme of the analytical strategy of the OFF–ON dual aptassay.
Fluorophore-labeled aptamers specific to procalcitonin (PCT) or interleukin-6 (IL-6) are immobilized onto micromotors (MM) through π-π bonds between the nucleotide bases and the hexagonal cells of their graphene oxide (GO) outer layers, resulting in MM-AptPCT and MM-AptIL-6.
This immobilization leads to quenching of the fluorophores on the GO surface, representing the “OFF” stage. Upon interaction with their respective proteins, PCT or IL-6, the specific affinity binding causes a conformational change in the aptamer structure, leading to desorption from the GO MM surface and fluorescence recovery, indicating the “ON” stage. Characterization of GO/Ni/platinum nanoparticles (PtNPs) MM through scanning electron microscopy (SEM) and X-ray spectroscopy analysis (EDX) confirms successful electrosynthesis and functionalization with specific aptamers (Fig 2).
The MM exhibit a nearly conical shape with dimensions of 5 μm width and 10 μm length. EDX analysis demonstrates the presence of phosphorus and nitrogen, confirming DNA presence on the MM surface.
The loss of nitrogen and phosphorus after interaction with biomarkers was observed, yet the remaining content did not hinder reliable quantitative analysis of clinical samples.
EDX mapping also verifies homogeneous distribution of MM components (carbon, nickel, and platinum), indicating efficient MM electrosynthesis.
MM-based dual aptassay: the optimization
The dual on-the-fly aptassay necessitates meticulous optimization of variables involved in both OFF and ON stages.
Challenges include optimizing the number of micromotors and aptamer concentrations for each biomarker, as well as demonstrating reliable simultaneous determination of procalcitonin (PCT) and interleukin-6 (IL-6) in ultra-reduced clinical sample volumes.
Sensitive determination of both PCT and IL-6 in low neonatal sample volumes (2 μL) is crucial. The use of such low volumes is particularly challenging for IL-6 analysis, where higher volumes lead to diminished fluorescence signals. Analysis times are also critical, with 15 minutes found to be the optimal interaction time, balancing efficient biomarker recognition with minimized interference from oxygen bubbles generated during MM propulsion.
Figure S3 illustrates incubation controls for PCT and IL-6 under different conditions, highlighting the superior biorecognition yields achieved with MM bubble propulsion.
This propulsion method improves biomarker detection sensitivity and reduces nonspecific adsorption, demonstrating the efficacy of MM in enhancing fluid mixing and immunoassay efficiency on the microscale.
MM-based dual aptassay: the analytical performance
Cross-reactivity is a crucial issue in the dual assay. As can be observed in Fig. 3, for each assay, there are barely any differences between the signals obtained in the absence and presence of the other protein, demonstrating excellent assay selectivity during the
real-time dual assay where both MM are simultaneously navigating in just 2 μL of the same sample.
Cross-reactivity for dual aptassay of PCT (A) and IL-6 (B). For PCT: a) 0 PCT; b) 0 PCT + 2 μg/mL IL-6; c) 3 μg/mL PCT; d) 3 μg/mL PCT + 2 μg/mL IL-6. For IL-6: a) 0 IL-6; b) 0 IL-6 + 3 μg/mL PCT; c) 2 μg/mL IL-6; d) 2 μg/mL IL-6 + 3 μg/mL PCT.
MM-based dual aptassay: clinical sample analysis
Our dual PCT and IL-6 aptassay, utilizing MM technology, represents a pioneering approach, showcasing reliable simultaneous determination of both biomarkers in the same clinical sample without compromising analytical performance.
Compared to single determinations, our method exhibits improved sensitivity, reduced sample volume (only 2 μL), and faster analysis times (15 min).
In comparison with other analytical approaches for simultaneous determination of PCT and IL-6, our MM-aptassay-based approach proves highly competitive, achieving better sensitivities, faster analysis times, and requiring smaller sample volumes.
Additionally, our analysis of real neonatal samples adds exceptional value to the results, demonstrating the promising potential of MM technology, despite being in its adolescent stage.
The proposed approach offers distinct technical advantages, including minimal sample volume requirements and rapid results, enabling real-time monitoring of sepsis biomarkers in high-risk neonates.
This capability may facilitate prompt and informed decisions about the initiation of antibiotic therapy, crucial in managing sepsis in neonates.
Conclusion
The development of a fluorescence dual micromotor (MM)-based aptassay allows for simultaneous, rapid, and accurate determination of key biomarkers like procalcitonin (PCT) and interleukin-6 (IL-6) in clinical samples from neonates with gestational age less than 32 weeks and birthweight below 1000 g suspected of late-onset sepsis.
This breakthrough underscores MM technology’s potential as a competitive tool in sepsis diagnosis, paving the way for innovative health initiatives. By embracing cutting-edge technology, we aim to revolutionize the paradigm of sepsis identification, analysis, and dissemination of new diagnostic technologies. The results demonstrate the analytical capabilities of MM technology in ultra-miniaturized clinical neonatal samples, offering multiplexing analysis for assessing multiple proteins throughout the diagnostic window of sepsis.
This advancement opens avenues for bedside/point-of-care tools for diagnosing sepsis in critically ill patients, especially when blood sample availability is limited, and timely recognition is critical. While MM technology in diagnosing neonatal sepsis is promising, it requires further research.
Source: Gordón Pidal, J.M., Arruza, L., Moreno-Guzmán, M. et al. Micromotor-based dual aptassay for early cost-effective diagnosis of neonatal sepsis. Microchim Acta 191, 106 (2024). https://doi.org/10.1007/s00604-023-06134-x
