A microwell-based impedance sensor on an insertable microneedle for real-time in vivo cytokine detection

AbstractImpedance-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.

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Distinguishing dopamine and calcium responses using XNA-nanotube sensors for improved neurochemical sensing

10.1101/2021.02.20.428669 ◽

2021 ◽

Author(s):

Alice J. Gillen ◽

Alessandra Antonucci ◽

Melania Reggente ◽

Daniel Morales ◽

Ardemis A. Boghossian

Keyword(s):

Neurological Diseases ◽

Locked Nucleic Acid ◽

Optical Biosensors ◽

Label Free ◽

Sensing Applications ◽

Label Free Detection ◽

Improved Stability ◽

New Applications

AbstractTo date, the engineering of single-stranded DNA-SWCNT (DNA-SWCNT) optical biosensors have largely focused on creating sensors for new applications with little focus on optimising existing sensors for in vitro and in vivo conditions. Recent studies have shown that nanotube fluorescence can be severely impacted by changes in local cation concentrations. This is particularly problematic for neurotransmitter sensing applications as spatial and temporal fluctuations in the concentration of cations, such as Na+, K+, or Ca2+, play a central role in neuromodulation. This can lead to inaccuracies in the determination of neurotransmitter concentrations using DNA-SWCNT sensors, which limits their use for detecting and treating neurological diseases.Herein, we present new approaches using locked nucleic acid (LNA) to engineer SWCNT sensors with improved stability towards cation-induced fluorescence changes. By incorporating LNA bases into the (GT)15-DNA sequence, we create sensors that are not only more resistant towards undesirable fluorescence modulation in the presence of Ca2+ but that also retain their capabilities for the label-free detection of dopamine. The synthetic biology approach presented in this work therefore serves as a complementary means for enhancing nanotube optoelectronic behavior, unlocking previously unexplored possibilities for developing nano-bioengineered sensors with augmented capabilities.

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A transgenic reporter mouse model for in vivo assessment of retinoic acid receptor transcriptional activation

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000705 ◽

2021 ◽

pp. 1-13

Author(s):

Harald Carlsen ◽

Kanae Ebihara ◽

Nobuyo H. Kuwata ◽

Kazuhisa Kuwata ◽

Gamze Aydemir ◽

...

Keyword(s):

Retinoic Acid ◽

Vitamin A ◽

Transgenic Mice ◽

Real Time ◽

Luciferase Activity ◽

Retinoic Acid Receptor ◽

Acid Receptor ◽

Deficient Mice ◽

In Vivo Assessment

Abstract. Background: Vitamin A is essential for a wide range of life processes throughout embryogenesis to adult life. With the aim of developing an in vivo model to monitor retinoic acid receptor (RAR) transactivation real-time in intact animals, we generated transgenic mice carrying a luciferase (luc) reporter gene under the control of retinoic acid response elements (RAREs) consisting of three copies of a direct repeat with five spacing nucleotides (DR5). Methods: Transgenic mice carrying a RARE dependent luciferase reporter flanked with insulator sequence were generated by pronuclear injection. RARE dependent luciferase activity was detected by in vivo imaging or in tissue extracts following manipulations with RAR/retinoid X receptor (RXR) agonists, RAR antagonists or in vitamin A deficient mice. Results: We found a strong induction of luciferase activity in a time and dose dependent manner by retinoic acid as well as RAR agonists, but not by the RXR agonist (using n=4–6 per group; 94 mice). In addition, luciferase activity was strongly reduced in vitamin A-deficient mice (n=6–9; 30 mice). These observations confirm that luciferase activity was controlled by RAR activation in the RARE-luc mouse. Luciferase activity was detectable in various organs, with high activity especially in brain and testis, indicating strong retinoid signalling in these tissues. Conclusion: The RARE-luc transgenic mice, which enabled real-time in vivo assessment of RAR activation, will be useful in understanding the normal physiology of vitamin A, the role of retinoid signalling in pathologies as well as to evaluate pharmacological ligands for RARs.

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Electrical Porous Silicon Microarray for dna Hybridization Detection

MRS Proceedings ◽

10.1557/proc-782-a7.2 ◽

2003 ◽

Vol 782 ◽

Cited By ~ 3

Author(s):

Marie Archer ◽

Marc Christophersen ◽

Philippe M. Fauchet ◽

Deoram Persaud ◽

Karl D. Hirschman

Keyword(s):

Porous Silicon ◽

Real Time ◽

Dna Hybridization ◽

Electrical Contacts ◽

Macroporous Silicon ◽

Label Free ◽

Internal Surface Area ◽

Free Standing ◽

Sensing Applications ◽

Label Free Detection

ABSTRACTThe sensitivity of Porous Silicon (PSi) to the presence of charged molecules and its large internal surface area represent two important properties that make this material and ideal candidate for electrical biosensor development. We have demonstrated the use of a macroporous silicon electrical sensor for label-free detection of DNA hybridization in real time as well as identification of organic solvents in liquid phase. Binding of DNA inside the PSi matrix induces a change in capacitance and conductance. Having demonstrated the suitability of macroporous silicon layers for real time detection of DNA hybridization on single devices, we have extended our findings to the fabrication of a microarray with individual device electrical addressing capabilities. On a crystalline p-type silicon wafer, process steps such as KOH etching and electrochemical dissolution are employed in selected regions to create a free-standing porous membrane for sensing applications. Individual electrical contacts are made on the front side of the wafer while the infiltration of the probe and target molecules is done from the back avoiding any direct interaction of the molecules with the contact sites. We will report on the design considerations of the electrical porous silicon array and the preliminary results obtained using synthetic DNA as a model molecule.

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