Combining the specificity of tumor‐targeting bacteria with the sensitivity of biomarker detection would create a screening method able to detect small tumors and metastases. To create this system, we genetically modified an attenuated strain of
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- NSF-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
More Like this
-
more » « less
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. -
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.more » « less
-
Quantitative and dynamic analyses of immune cell secretory cytokines are essential for precise determination and characterization of the “immune phenotype” of patients for clinical diagnosis and treatment of immune-related diseases. Although multiple methods including the enzyme-linked immunosorbent assay (ELISA) have been applied for cytokine detection, such measurements remain very challenging in real-time, high-throughput, and high-sensitivity immune cell analysis. In this paper, we report a highly integrated microfluidic device that allows for on-chip isolation, culture, and stimulation, as well as sensitive and dynamic cytokine profiling of immune cells. Such a microfluidic sensing chip is integrated with cytometric fluorescent microbeads for real-time and multiplexed monitoring of immune cell cytokine secretion dynamics, consuming a relatively small extracted sample volume (160 nl) without interrupting the immune cell culture. Furthermore, it is integrated with a Taylor dispersion-based mixing unit in each detection chamber that shortens the immunoassay period down to less than 30 minutes. We demonstrate the profiling of multiple pro-inflammatory cytokine secretions ( e.g. interleukin-6, interleukin-8, and tumor necrosis factors) of human peripheral blood mononuclear cells (PBMCs) with a sensitivity of 20 pg ml −1 and a sample volume of 160 nl per detection. Further applications of this automated, rapid, and high-throughput microfluidic immunophenotyping platform can help unleash the mechanisms of systemic immune responses, and enable efficient assessments of the pathologic immune status for clinical diagnosis and immune therapy.more » « less
-
more » « less
Neurotoxic heavy metals, such as Cd2+, pose a significant global health concern due to their increased environmental contamination and subsequent detrimental health hazards they pose to human beings. These metal ions can breach the blood-brain barrierblood–brain barrier, leading to severe and often irreversible damage to the central nervous system and other vital organs. Therefore, developing a highly sensitive, robust, and rapid in vivo detection method for these hazardous heavy metal ions is of the utmost importance for early detection, thus initiating timely therapeutics. Detecting ultra-low levels of toxic metal ions in vivo and obtaining accurate speciation information remains a challenge with conventional analytical techniques. In this study, we fabricated a novel carbon carbon-fiber microelectrode (CFM)-based sensor that can detect Cd2+ ions using fast-scan cyclic voltammetry by electrodepositing gold nanoparticles (AuNP). We optimized electrochemical parameters that generate a unique cyclic voltammogram (CV) of Cd2+ at a temporal resolution of 100 ms with our novel sensor. All our experiments were performed in tris buffer that mimics the artificial cerebellum fluid. We established a calibration curve resulting in a limit of detection (LOD) of 0.01 µM with a corresponding sensitivity of 418.02 nA/ µM. The sensor’s selectivity was evaluated in the presence of other metal ions, and it was noteworthy to observe that the sensor retained its ability to produce the distinctive Cd2+ CV, even when the concentration of other metal ions was 200 times higher than that of Cd2+. We also found that our sensor could detect free Cd2+ ions in the presence of complexing agents. Furthermore, we analyzed the solution chemistry of each of those Cd2+–ligand solutions using a geochemical model, PHREEQC. The concentrations of free Cd2+ ions determined through our electrochemical data align well with geochemical modeling data, thus validating the response of our novel sensor. Furthermore, we reassessed our sensor’s LOD in tris buffer based on the concentration of free Cd2+ ions determined through PHREEQC analysis, revealing an LOD of 0.00132 µM. We also demonstrated the capability of our sensor to detect Cd2+ ions in artificial urine samples, showcasing its potential for application in actual biological samples. To the best of our knowledge, this is the first AuNP-modified, CFM-based Cd2+ sensor capable of detecting ultra-low concentrations of free Cd2+ ions in different complex matrices, including artificial urine at a temporal resolution of 100 ms, making it an excellent analytical tool for future real-time, in vivo detection, particularly in the brain.
-
more » « less
Abstract Antibody detection assays are essential for evaluating immunity of individuals against a given virus, and this has been particularly relevant during the COVID‐19 pandemic. Current serology assays either require a laboratory setting and take >1 hr (i.e., enzyme‐linked immunosorbent assay [ELISA]) or are rapid but only qualitative in nature and cannot accurately track antibody levels over time (i.e., lateral flow assay [LFA]). Therefore, there is a need for development of a rapid and simple but also quantitative assay that can evaluate antibody levels in patients accurately over time. We have developed an assay that uses a split nanoluciferase fused to the spike or nucleocapsid proteins of the SARS‐CoV‐2 virus to enable luminescent‐based detection of spike‐ or nucleocapsid‐binding antibodies in serum, plasma, and whole blood samples. The resulting approach is simple, rapid, and quantitative and is highly amenable to low‐/medium‐throughput scale using plate‐based assays, high‐throughput scale using robotics, and point‐of‐care applications. In this article, we describe how to perform the assay in a laboratory setting using a plate reader or liquid‐handling robotics and in a point‐of‐care setting using a handheld, battery‐powered luminometer. Together, these assays allow antibody detection to be easily performed in multiple settings by simplifying and reducing assay time in a laboratory or clinical environment and by allowing for antibody detection in point‐of‐care, nonlaboratory settings. © 2022 Wiley Periodicals LLC.
Basic Protocol : SARS‐CoV‐2 antibody detection using the split‐luciferase assay on a medium‐throughput scale with a laboratory luminometerAlternate Protocol 1 : High‐throughput‐based protocol for SARS‐CoV‐2 antibody detection using a robotic platformAlternate Protocol 2 : Point‐of‐care‐based protocol for SARS‐CoV‐2 antibody detection using a handheld luminometerSupport Protocol : Determining positive/negative cutoffs for test samples and standardizing the assay between days
