The inflammation marker Interleukin 6 (IL-6) typically remains below 5 pg/mL in the serum of healthy individuals but can increase tenfold during inflammation in chronic conditions like COVID-19 and rheumatoid arthritis, as well as acute conditions like sepsis. This study is focused on the rapid detection of IL-6 to monitor both chronic and acute diseases. The novel sensor, designed with gold-coated micropyramids on the electrodes, was fabricated using the two-photon polymerization method, enabling low-volume sensing capabilities (2-3 μL). The micropyramids were surface functionalized with interleukin-6 antibodies towards developing an affinity biosensor specific to the physiological relevant range of IL-6 of 5.1 and 18.8 pg/mL in mild inflammation. Sensing was achieved by measuring impedance changes associated with IL-6 binding to the antibodies on the micropyramids interfaced using electrochemical impedance spectroscopy. It was observed that the signals from the lowest detection concentration was enhanced by 3 times at 1500 hz when the 532 nm green laser was incident on the micropyramids. This innovative approach can be expanded to the detection of cytokines not only in serum but also in respiratory samples. As a result, it opens up new avenues for monitoring local inflammation within the lungs and assessing systemic inflammation levels throughout the body.
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A microwell-based impedance sensor on an insertable microneedle for real-time in vivo cytokine detection
Impedance-based protein detection sensors for point-of-care diagnostics require quantitative specificity, as well as rapid or real-time operation. Furthermore, microfabrication of these sensors can lead to the formation of factors suitable for in vivo operation. Herein, we present microfabricated needle-shaped microwell impedance sensors for rapid-sample-to-answer, label-free detection of cytokines, and other biomarkers. The microneedle form factor allows sensors to be utilized in transcutaneous or transvascular sensing applications. In vitro, experimental characterization confirmed sensor specificity and sensitivity to multiple proteins of interest. Mechanical characterization demonstrated sufficient microneedle robustness for transcutaneous insertion, as well as preserved sensor function postinsertion. We further utilized these sensors to carry out real-time in vivo quantification of human interleukin 8 (hIL8) concentration levels in the blood of transgenic mice that endogenously express hIL8. To assess sensor functionality, hIL8 concentration levels in serum samples from the same mice were quantified by ELISA. Excellent agreement between real-time in vivo sensor readings in blood and subsequent ELISA serum assays was observed over multiple transgenic mice expressing hIL8 concentrations from 62 pg/mL to 539 ng/mL.
more » « less- PAR ID:
- 10360382
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Microsystems & Nanoengineering
- Volume:
- 7
- Issue:
- 1
- ISSN:
- 2055-7434
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Salmonella enterica to release a recombinant fluorescent biomarker (or fluoromarker).Salmonella expressing ZsGreen were intravenously administered to tumor‐bearing mice and fluoromarker production was induced after 48 hr. The quantities and locations of bacteria and ZsGreen were measured in tumors, livers and spleens by immunofluorescence, and the plasma concentration of ZsGreen was measured using single‐layer ELISA. In the plasma, the ZsGreen concentration was in the range of 0.5–1.5 ng/ml and was dependent on tumor mass (with a proportion of 0.81 ± 0.32 ng·ml−1·g−1). No adverse reaction to ZsGreen or bacteria was observed in any mice. ZsGreen was released at an average rate of 4.3 fg·CFU−1·hr−1and cleared from the plasma with a rate constant of 0.259 hr−1. ZsGreen production was highest in viable tissue (7.6 fg·CFU−1·hr−1) and lowest in necrotic tissue (0.47 fg·CFU−1·hr−1). The mass transfer rate constant from tumor to blood was 0.0125 hr−1. Based on these measurements, this system has the capability to detect tumors as small as 0.12 g. These results demonstrate four essential mechanisms of this method: (i ) preferential tumor colonization by bacteria, (ii ) fluoromarker releasein vivo , (iii ) fluoromarker transport through tumor tissue and (iv ) slow enough systemic clearance to enable measurement. This bacteria‐based blood test would be minimally invasive and has the potential to identify previously undetectable microscopic tumors. -
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Colorimetric enzyme-linked immunosorbent assay (ELISA) has been widely applied as the gold-standard method for cytokine detection for decades. However, it has become a critical challenge to further improve the detection sensitivity of ELISA, as it is limited by the catalytic activity of enzymes. Herein, we report an enhanced colorimetric ELISA for ultrasensitive detection of interleukin-6 (IL-6, as a model cytokine for demonstration) using Pd@Pt core@shell nanodendrites (Pd@Pt NDs) as peroxidase nanomimics (named “Pd@Pt ND ELISA”), pushing the sensitivity up to femtomolar level. Specifically, the Pd@Pt NDs are rationally engineered by depositing Pt atoms on Pd nanocubes (NCs) to generate rough dendrite-like Pt skins on the Pd surfaces via Volmer–Weber growth mode. They can be produced on a large scale with highly uniform size, shape, composition, and structure. They exhibit significantly enhanced peroxidase-like catalytic activity with catalytic constants (Kcat) more than 2000-fold higher than those of horseradish peroxidase (HRP, an enzyme commonly used in ELISA). Using Pd@Pt NDs as the signal labels, the Pd@Pt ND ELISA presents strong colorimetric signals for the quantitative determination of IL-6 with a wide dynamic range of 0.05–100 pg mL−1 and an ultralow detection limit of 0.044 pg mL−1 (1.7 fM). This detection limit is 21-fold lower than that of conventional HRP-based ELISA. The reproducibility and specificity of the Pd@Pt ND ELISA are excellent. More significantly, the Pd@Pt ND ELISA was validated for analyzing IL-6 in human serum samples with high accuracy and reliability through recovery tests. Our results demonstrate that the colorimetric Pd@Pt ND ELISA is a promising biosensing tool for ultrasensitive determination of cytokines and thus is expected to be applied in a variety of clinical diagnoses and fundamental biomedical studies.more » « less
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